Application of intracellular nanovesicles derived from mesenchymal stem cells in neuroprotection

Abstract

The present invention discloses an application of intracellular nanovesicles derived from mesenchymal stem cells in neuroprotection. Compared with small extracellular vesicles with exosomes as the main component, the small intracellular nanovesicles of the present invention have a smaller particle size, a narrow particle size distribution range, are more stable at different temperatures, and have good tissue compatibility. The small intracellular nanovesicles of the present invention can better improve nerve damage or neurodegenerative diseases such as optic nerve damage, ischemic stroke, Alzheimer's disease, etc., and have very good application and research value in the field of medicine.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of intracellular nanovesicles derived from mesenchymal stem cells in neuroprotection, in particular to an application of intracellular nanovesicles derived from mesenchymal stem cells in the treatment of nerve damage and neurodegeneration. Background Art

[0002] The nervous system is a system that plays a leading role in regulating physiological functions in the body. It is mainly composed of nerve tissue and is divided into two parts: the central nervous system and the peripheral nervous system. The central nervous system (CNS) includes the brain and spinal cord. Its neurons usually cannot regenerate after injury, resulting in loss of nerve function. Brain nerve damage mainly leads to neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, Huntington's disease, etc.), traumatic brain injury and cerebral ischemia, while spinal cord injury often causes patients to have obvious limb sensory and motor dysfunction. The optic nerve is usually considered to be part of the central nervous system. It is composed of axons of retinal ganglion cells (RGCs) and can transmit visual information collected by the retina to the cerebral cortex. Glaucoma, eye trauma, tumor lesions, and drug poisoning can all cause damage to the optic nerve. Once the optic nerve is damaged, it often causes decreased vision, weakened color vision, and even loss of vision. Given the non-regenerative nature of central nervous system neurons and the severe functional impairment after nerve injury, finding new neurotherapeutic strategies is of great significance for exploring the mechanism of central nervous system injury repair and promoting central nervous system regeneration.

[0003] Mesenchymal stem cells (MSCs) cell therapy has become a promising treatment for neurological diseases. This treatment method depends on the ability of mesenchymal stem cells to transdifferentiate into neural cells, as well as their self-renewal potential, pro-proliferation properties and neuroprotective effects. However, there are many bottlenecks in the application of cell therapy in neurological diseases, such as abnormal cell growth and immune rejection. Studies have found that extracellular vesicles (EVs) secreted by mesenchymal stem cells are believed to be able to replace cells to play a therapeutic role because of their stem cell properties, but they also have certain defects, such as low efficiency of extracellular vesicle collection, extracellular vesicles are present in the extracellular matrix after being secreted, and they contain exogenous substances in the cell culture medium, resulting in reduced purity.

[0004] It is worth noting that there are many nanoscale vesicles in the cell, which are located between various membrane-rich organelles and are responsible for the intracellular material transport and secretory pathways. These intracellular vesicles (IVs) are produced by a process called vesicle budding and can originate from various organelles, including the endoplasmic reticulum, Golgi apparatus, endosomes, and plasma membranes. They are composed of a variety of membrane-rich particles, such as constitutive secretory vesicles, synaptic vesicles, COP-coated vesicles, Golgi-derived vesicles, clathrin-coated vesicles, and transport vesicles between the endoplasmic reticulum and the Golgi apparatus. These IVs maintain the basic life activities in the cell, which contain a large number of biological macromolecules, and participate in the secretion of specific proteins, hormones, and other biological molecules through exocytosis.

[0005] The inventors of the present invention have found that these intracellular nanovesicles can not only replace cells to play a therapeutic role, but also have potential application value in neuroprotection. However, previous studies have mainly focused on the signal transmission and regulation of intracellular nanovesicle-mediated substance transport, and there are no reports in the prior art on the use of intracellular nanovesicles for neuroprotection. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the present invention provides an application of intracellular nanovesicles derived from mesenchymal stem cells in neuroprotection (particularly in the protection of nerve damage and neurodegeneration).

[0007] In a first aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating nervous system diseases.

[0008] Specifically, the vesicles are small intracellular nanovesicles (sIVs).

[0009] Specifically, the vesicle is double-layered horseshoe-shaped or teacup-shaped.

[0010] Specifically, the average particle size of the vesicles is 50-100 nm (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 nm), in particular 65-85 nm.

[0011] Specifically, the vesicles express TMEM214 protein.

[0012] Specifically, the vesicles low-express exosome markers and high-express marker proteins of intracellular membrane-rich organelles and the Clathrin protein family.

[0013] Specifically, the vesicles are relatively stable at -80°C to 37°C (e.g., -80, -50, -20, -10, 0, 5, 25, 37°C).

[0014] Specifically, the vesicles are prepared by a method comprising an ultrasonic treatment step. More specifically, the vesicles are prepared by a method comprising an ultrasonic treatment, a centrifugation treatment, and an ultracentrifugation treatment step (performed sequentially).

[0015] In a preferred embodiment of the present invention, the vesicles are prepared by a method comprising the following steps:

[0016] (1) Dispersing mesenchymal stem cells in a suspension solvent and subjecting them to ultrasonic treatment;

[0017] (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding cell membranes and organelle fragments, and taking the supernatant;

[0018] (3) subjecting the supernatant obtained in step (2) to ultracentrifugation to obtain the precipitate as intracellular nanovesicles;

[0019] Optionally, (4) resuspending the precipitate obtained in step (3).

[0020] Specifically, the cells in step (1) are cells isolated after culture, digestion and washing (after discarding the cell culture medium, digestion, washing and other steps, the possibility of isolating and obtaining extracellular vesicles can be eliminated).

[0021] Specifically, the method may further include cell digestion and counting steps; in some embodiments of the present invention, the cell digestion step includes: culturing cells to grow to 90% confluence, discarding the cell culture medium, washing the cells, adding trypsin to digest the cells, and then neutralizing and washing the cells.

[0022] Specifically, the cell density of the cells in the suspension solvent is 1-4×10 6 / mL, for example 1×10 6 / mL, 2×10 6 / mL, 3×10 6 / mL, 4×10 6 In some embodiments of the present invention, the cell density is 1×10 6 Pieces / mL.

[0023] Specifically, the suspension solvent is any buffer suitable for culturing cells, such as PBS, Tris buffer, glycine buffer. In some embodiments of the present invention, the solvent is PBS.

[0024] Specifically, the mesenchymal stem cells are derived from mammals, especially humans.

[0025] Specifically, the mesenchymal stem cells are selected from: umbilical cord mesenchymal stem cells (UC-MSC), bone marrow mesenchymal stem cells (BM-MSC), adipose mesenchymal stem cells (AD-MSC), dental pulp mesenchymal stem cells, placenta and amniotic fluid and amniotic membrane mesenchymal stem cells, especially umbilical cord mesenchymal stem cells (UC-MSC).

[0026] Specifically, the amplitude of the ultrasonic treatment in step (1) is 20%-35% (e.g., 20%, 22%, 24%, 25%, 30%, 35%), preferably 20%-25%. In some embodiments of the present invention, the amplitude of the ultrasonic treatment is 20%.

[0027] Specifically, the ultrasonic treatment time in step (1) is 15-60s (e.g., 15, 20, 25, 30, 40, 50, 60s), preferably 15s. In some embodiments of the present invention, the ultrasonic treatment time is 15s, on 2s, off 2s.

[0028] Specifically, the ultrasonic treatment time in step (1) is 10-20 s (e.g., 10, 15, 18, 20 s).

[0029] In some embodiments of the present invention, the number of centrifugation treatments in step (2) is two times, and the respective parameters are:

[0030] 1000-3000 g (e.g. 1000, 1500, 2000, 2500, 3000 g), 5-20 minutes (e.g. 5, 8, 10, 12, 15, 20 minutes);

[0031] 10000-30000 g (e.g. 10000, 15000, 20000, 25000, 30000 g), 20-40 min (e.g. 20, 25, 28, 30, 32, 35, 40 min).

[0032] In one embodiment of the invention, the first centrifugation is performed at 2000 g for 10 minutes.

[0033] In one embodiment of the invention, the second centrifugation is performed at 20,000 g for 30 minutes.

[0034] Specifically, the parameters of the ultracentrifugation treatment in step (3) include 100,000-180,000 g (e.g., 100,000, 120,000, 140,000, 150,000, 160,000, 180,000 g), 50-100 minutes (e.g., 50, 60, 65, 70, 75, 80, 90, 100 minutes).

[0035] In one embodiment of the invention, the ultracentrifugation is performed at 150000 g for 70 minutes.

[0036] Specifically, the resuspension solvent in step (4) is any buffer suitable for culturing cells, such as PBS, Tris buffer, glycine buffer. In some embodiments of the present invention, the resuspension solvent is PBS.

[0037] Specifically, one or more of the ultrasonic treatment, centrifugation treatment, and ultracentrifugation treatment are performed at low temperature, such as 0-5°C; in particular, the ultrasonic treatment, centrifugation treatment, and ultracentrifugation treatment are all performed on ice; or, the ultrasonic treatment, centrifugation treatment, and ultracentrifugation treatment are all performed at 4°C.

[0038] In some embodiments of the present invention, the vesicles are prepared by a method comprising the following steps: taking 1×10 6 For a cell suspension with a density of 1 cell / mL, place the ultrasonic probe in the center of the liquid surface, and perform ultrasonic treatment with an ultrasonic amplitude parameter range of 20%, a time parameter range of 15s, on (run) 2s, off (pause) 2s; then transfer the liquid to a centrifuge tube for centrifugation with centrifugation parameters of 2000g×10min, 20000g×30min, and collect the supernatant; transfer the supernatant to an ultracentrifuge tube for centrifugation with centrifugation parameters of 150000g×70min.

[0039] In some embodiments of the present invention, the nervous system disease is nerve damage and nerve degeneration and related diseases.

[0040] Specifically, the nerve damage can be nerve damage caused by various reasons, for example, infection (such as viruses, bacteria, fungi, parasites, etc.), trauma (such as chemical damage, photochemical damage, knife cuts, burns, firearm injuries, abrasions, extrusions, impacts, etc.), compression or traction injuries (such as intervertebral disc herniation, spinal stenosis, brachial plexus injury, etc.), autoimmune mechanisms (such as myasthenia gravis, multiple sclerosis, etc.), ischemic injuries (such as stroke, cerebral thrombosis, etc.), metabolic diseases (such as diabetes, etc.), tumors, hereditary, nutritional, inflammation, drug damage, and its manifestations may include, but are not limited to, movement disorders (such as muscle weakness, paralysis or tremor, etc.), sensory disorders (such as numbness, tingling, paresthesia or loss of sensation, etc.), autonomic nerve disorders (such as hypotension, syncope, constipation or urinary incontinence, etc.), cognitive disorders (such as amnesia, confusion or inattention, etc.), mental disorders (such as depression, anxiety, abnormal behavior or mental confusion, etc.).

[0041] Specifically, the nerve injury-related diseases include, but are not limited to, spinal cord diseases, optic nerve diseases, cerebrovascular diseases, traumatic brain injury, extrapyramidal diseases, demyelinating diseases, neurodegenerative diseases, central nervous system infectious diseases, cerebral palsy, hydrocephalus, cranial nerve diseases, nerve root diseases, plexus diseases, single nerve diseases, multiple nerve diseases, neuromuscular junction diseases, ataxia, leukodystrophy, mental retardation, familial amyloid polyneuropathy, Charcot-Marie-Tooth disease, and neurosis.

[0042] Specifically, the neurodegeneration-related diseases include, but are not limited to, motor neuron disease (such as amyotrophic lateral sclerosis, progressive spinal muscular atrophy, pyramidal tract degeneration, progressive bulbar palsy, pseudobulbar palsy, true bulbar palsy, primary lateral sclerosis, spinobulbar muscular atrophy, lower motor neuron syndrome, upper motor neuron syndrome, flail arm syndrome, Madras motor neuron disease), Alzheimer's disease, multiple system atrophy, senile brain atrophy, Pick's disease dementia, alcoholic nervous system degeneration (such as alcoholic cerebellar degeneration, alcoholic polyneuropathy, alcoholic myopathy, central pontine myelinolysis, corpus callosum degeneration, alcoholic dementia, alcoholic mental disorder), frontotemporal dementia, subacute necrotizing encephalopathy, corticostriatal spinal degeneration, Lewy body dementia, Machado-Joseph disease.

[0043] Specifically, the spinal cord disease is selected from: spinal cord injury, myelitis, poliomyelitis, encephalomyelitis, spinal cord tumor, spinal muscular atrophy, motor neuron disease, spinal cord compression, syringomyelia, syringomyelia, vascular myelopathy (such as spinal cord hemorrhage, spinal cord ischemia, spinal cord embolism, spinal cord infarction, spinal cord necrosis, spinal cord edema, spinal artery thrombosis, posterior spinal artery syndrome, anterior spinal artery occlusive syndrome, spinal vascular malformation, spinal arteriovenous malformation), hepatic myelopathy, cervical spondylotic myelopathy, spinal meningitis, epidural abscess, spinal disease, cauda equina syndrome, HTLV-1 associated myelopathy / tropical spastic paralysis.

[0044] Specifically, the optic nerve disease is selected from: optic nerve tumors, ischemic optic neuropathy, hereditary optic neuropathy, compressive optic neuropathy, optic nerve injury, nutritional optic neuropathy, toxic optic neuropathy, optic neuritis, papilledema, optic nerve atrophy, and glaucoma.

[0045] Specifically, the cerebrovascular disease is selected from: ischemic cerebrovascular disease (such as cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism, ischemic hypoxic encephalopathy), hemorrhagic cerebrovascular disease (such as cerebral hemorrhage, subarachnoid hemorrhage, non-traumatic subdural hemorrhage, non-traumatic epidural hemorrhage), cerebral atherosclerosis, cerebral arteritis, cerebral aneurysm, cerebral arteriovenous fistula, amyloid cerebrovascular disease, intracranial venous thrombosis, intracranial blood pressure malformation (such as cerebral vascular arteriovenous malformation, cerebral cavernous malformation, dural arteriovenous fistula, carotid cavernous sinus fistula), intracranial telangiectasia, reversible cerebral vasoconstriction syndrome, vascular dementia (such as Binswanger's disease, multiple cerebral infarction dementia, hemorrhagic dementia), hypertensive encephalopathy, moyamoya disease, red core syndrome, Weber syndrome, Benedict's syndrome, vascular leukoencephalopathy.

[0046] Specifically, the extrapyramidal disease is selected from: Hallevorden-Spatz disease, progressive supranuclear palsy, multiple system atrophy, calcification of the basal ganglia, dystonia, orofacial dyskinesia, Meige syndrome, essential tremor, myoclonus, chorea, restless legs syndrome, stiff-person syndrome, and Wilson's disease.

[0047] Specifically, the demyelinating disease is selected from: multiple sclerosis (such as brainstem multiple sclerosis, spinal cord multiple sclerosis, multiple sclerosis dementia), acute disseminated encephalomyelitis, neuromyelitis optica, acute hemorrhagic leukoencephalitis, diffuse sclerosis, central pontine myelinolysis, extrapontine myelinolysis, acute transverse myelitis, subacute necrotizing myelitis, concentric sclerosis, tumor-like demyelinating lesions, cerebral white matter demyelination, demyelinating myelitis, and clinically isolated syndrome.

[0048] Specifically, the neurodegenerative disease is selected from the group consisting of epilepsy, cerebral ischemia, brain injury, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, ataxia-telangiectasia syndrome, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, multiple sclerosis, primary lateral sclerosis, spinal muscular atrophy, and spastic paraplegia.

[0049] Specifically, the central nervous system infectious disease is selected from: encephalitis, meningitis (such as pachymeningitis, piameningitis, arachnoiditis), myelitis, encephalomyelitis, ventriculitis, cerebellitis, brainstem inflammation, intracranial abscess (such as brain abscess, epidural abscess, subdural abscess), intracranial granuloma (such as brain granuloma, epidural granuloma, subdural granuloma), neurosyphilis, brain parasitic disease, prion disease.

[0050] Specifically, the cerebral palsy is selected from: spastic cerebral palsy, dyskinetic cerebral palsy, ataxic cerebral palsy, and Worster-Drought syndrome.

[0051] Specifically, the hydrocephalus is selected from: communicating hydrocephalus, obstructive hydrocephalus, compensatory hydrocephalus, and congenital hydrocephalus.

[0052] Specifically, the cranial nerve disease is selected from: trigeminal nerve disease (such as trigeminal neuralgia), facial nerve disease (such as Bell's palsy, geniculate ganglionitis, Merrow syndrome, hemifacial spasm, hemifacial atrophy, facial neuritis), olfactory nerve disease, glossopharyngeal nerve disease, vagus nerve disease, hypoglossal nerve disease, multiple cranial nerve diseases.

[0053] Specifically, the nerve root disease is selected from: nerve compression, nerve root sleeve cyst.

[0054] Specifically, the plexus disease is selected from: brachial plexus injury and lumbar plexus injury.

[0055] Specifically, the mononeuropathy is selected from: upper limb mononeuropathy (such as carpal tunnel syndrome, ulnar neuropathy, radial nerve damage), lower limb mononeuropathy (such as sciatic nerve damage, femoral neuropathy, tibial neuropathy, tarsal tunnel syndrome, plantar nerve damage, intercostal neuropathy).

[0056] Specifically, the polyneuropathy is selected from: idiopathic progressive neuropathy, inflammatory polyneuropathy, secondary polyneuropathy (such as diabetic polyneuropathy, infectious polyneuropathy, dystrophic polyneuropathy).

[0057] Specifically, the neuromuscular junction disease is selected from: myasthenia gravis, myasthenic syndrome, muscular dystrophy, myotonia-related diseases (such as neuromyotonia, atrophic myotonia, dystrophic myotonia, congenital myotonia, paramyotonia), mitochondrial myopathy, alcoholic myopathy, inflammatory myopathy, muscular atrophy, periodic paralysis, ocular myopathy, and myofibrillar myopathy.

[0058] Specifically, the ataxia is selected from: cerebellar ataxia (such as Marinesco-Sjogren syndrome, Ramsay-Hunt syndrome, paroxysmal ataxia, Friedreich ataxia, spinocerebellar ataxia, hereditary spastic ataxia, Kearn-Sayre syndrome, ataxia telangiectasia, CANVAS syndrome), hereditary spastic paraplegia.

[0059] Specifically, the neurosis is selected from the group consisting of hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, and somatoform disorder.

[0060] In some embodiments of the present invention, the nervous system disease is an optic nerve disease, for example, compressive optic neuropathy.

[0061] In some embodiments of the present invention, the nervous system disease is a cerebrovascular disease, for example, ischemic cerebrovascular disease, in particular cerebral ischemia.

[0062] In some embodiments of the present invention, the nervous system disease is a nervous system degenerative disease, in particular Alzheimer's disease or Parkinson's disease.

[0063] In some embodiments of the invention, the neurological disease is traumatic brain injury.

[0064] In some embodiments of the present invention, the nervous system disease is a spinal cord disease, in particular, a spinal cord injury.

[0065] Specifically, the nervous system disease is selected from: nerve damage disease and neurodegenerative disease.

[0066] Specifically, the nerve injury disease is selected from: optic nerve disease, ischemic cerebrovascular disease, traumatic brain injury and spinal cord injury.

[0067] Specifically, the neurodegenerative disease is selected from: Alzheimer's disease and Parkinson's disease.

[0068] In some embodiments of the present invention, the nerve damage disease is selected from: optic nerve damage, compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy, glaucoma, cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism and hypoxic-ischemic encephalopathy.

[0069] In some embodiments of the invention, the neurodegenerative disease is Alzheimer's disease.

[0070] Specifically, the drug can be administered in any suitable manner, including but not limited to oral administration, injection (such as intravenous injection, intramuscular injection, intraperitoneal injection), subcutaneous administration, skin administration, ocular administration (such as eye drops, eye ointment, subconjunctival injection, vitreous cavity injection), and nasal administration (such as nasal administration).

[0071] In one embodiment of the present invention, the administration method is ocular administration, particularly intravitreal injection.

[0072] In one embodiment of the present invention, the administration method is injection, particularly intravenous injection or intraperitoneal injection.

[0073] In one embodiment of the present invention, the administration method is nasal administration, especially nasal administration.

[0074] Specifically, the drug can be formulated into any suitable preparation form, such as, but not limited to, cream, foam, cream, ointment, emulsion, liquid solution, eye drops, injection, powder injection, gel, spray, suspension, microemulsion, eye mask or contact lens, etc., especially injection.

[0075] The present invention also provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating nerve damage diseases.

[0076] The present invention also provides a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of drugs for preventing and / or treating neurodegenerative diseases.

[0077] In a second aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a medicament for preventing and / or treating optic nerve damage, compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy or glaucoma.

[0078] Specifically, the intracellular nanovesicles derived from mesenchymal stem cells are as described in the first aspect of the present invention.

[0079] In a third aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating cerebral ischemia, cerebral infarction, cerebral thrombosis, cerebral embolism or ischemic-hypoxic encephalopathy.

[0080] Specifically, the intracellular nanovesicles derived from mesenchymal stem cells are as described in the first aspect of the present invention.

[0081] In a fourth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating Alzheimer's disease.

[0082] Specifically, the intracellular nanovesicles derived from mesenchymal stem cells are as described in the first aspect of the present invention.

[0083] In a fifth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating Parkinson's disease.

[0084] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0085] In a sixth aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating traumatic brain injury.

[0086] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0087] In a seventh aspect of the present invention, there is provided a use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of a drug for preventing and / or treating spinal cord diseases.

[0088] Specifically, the mesenchymal stem cell-derived intracellular nanovesicles are as described in the first aspect of the present invention.

[0089] Specifically, the spinal cord disease is spinal cord injury.

[0090] In an eighth aspect of the present invention, a method for preventing and / or treating a nervous system disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0091] Specifically, the nervous system disease is as described in the first aspect of the present invention.

[0092] Specifically, the subject is a mammal, especially a human.

[0093] Specifically, the administration can be carried out in any suitable manner, including but not limited to oral administration, injection (such as intravenous injection, intramuscular injection, intraperitoneal injection, subconjunctival injection, and vitreous cavity injection), subcutaneous administration, skin administration, ocular administration (such as intraocular administration, ocular surface administration, and periocular administration, specifically such as eye drops, eye ointment, subconjunctival injection, vitreous cavity injection, vitreous implantation, retrobulbar injection, peribulbar administration, subbulbar fascia administration, anterior chamber injection, and eye gel administration), and nasal administration (such as nasal administration).

[0094] Specifically, the dosage depends on many factors, including the subject's age, weight, sex, disease, severity, route and frequency of administration, etc., and therefore may vary. In some embodiments of the present invention, the dosage may be 1 μg-60 μg vesicle / eye, for example, 1 μg vesicle / eye, 2 μg vesicle / eye, 4 μg vesicle / eye, 5 μg vesicle / eye, 6 μg vesicle / eye, 8 μg vesicle / eye, 10 μg vesicle / eye, 15 μg vesicle / eye, 20 μg vesicle / eye, 25 μg vesicle / eye, 30 μg vesicle / eye, 35 μg vesicle / eye, 40 μg vesicle / eye, 45 μg vesicle / eye, 50 μg vesicle / eye, 55 μg vesicle / eye, 60 μg vesicle / eye.

[0095] In a ninth aspect of the present invention, a method for preventing and / or treating optic nerve damage is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof;

[0096] Alternatively, a method for preventing and / or treating compressive optic neuropathy, ischemic optic neuropathy, optic neuritis, optic atrophy or glaucoma is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0097] Specifically, the subject is a mammal, especially a human.

[0098] Specifically, the administration can be carried out by any suitable administration method, in particular, ocular administration (eg, intraocular administration, ocular surface administration), such as eye drops, eye ointment, subconjunctival injection, intravitreal injection, in particular intravitreal injection.

[0099] In some embodiments of the present invention, the administration method is intravitreal injection, and the administration amount can be 1 μg-30 μg vesicles / eye.

[0100] In a tenth aspect of the present invention, a method for preventing and / or treating cerebral ischemia is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof;

[0101] Alternatively, a method for preventing and / or treating cerebral infarction, cerebral thrombosis, cerebral embolism or hypoxic-ischemic encephalopathy is provided, which comprises the step of administering intracellular nanovesicles derived from mesenchymal stem cells (as described in the first aspect of the present invention) to a subject in need thereof.

[0102] Specifically, the subject is a mammal, especially a human.

[0103] Specifically, the administration can be carried out by any suitable administration method, in particular nasal administration, in particular nasal administration.

[0104] In an eleventh aspect of the present invention, a method for preventing and / or treating Alzheimer's disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0105] Specifically, the subject is a mammal, especially a human.

[0106] Specifically, the administration can be carried out by any suitable administration method, in particular nasal administration, in particular nasal administration.

[0107] In a twelfth aspect of the present invention, a method for preventing and / or treating Parkinson's disease is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0108] Specifically, the subject is a mammal, especially a human.

[0109] Specifically, the administration can be carried out by any suitable administration method, in particular nasal administration, in particular nasal administration.

[0110] In the thirteenth aspect of the present invention, a method for preventing and / or treating traumatic brain injury is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0111] Specifically, the subject is a mammal, especially a human.

[0112] Specifically, the administration can be carried out by any suitable administration method, in particular nasal administration, in particular nasal administration.

[0113] In a fourteenth aspect of the present invention, a method for preventing and / or treating spinal cord injury is provided, comprising the step of administering mesenchymal stem cell-derived intracellular nanovesicles (as described in the first aspect of the present invention) to a subject in need thereof.

[0114] Specifically, the subject is a mammal, especially a human.

[0115] Specifically, the administration can be carried out in any suitable manner, in particular by injection, for example, intravenous injection, intramuscular injection, intraperitoneal injection, in particular intravenous injection.

[0116] The present invention has the following excellent effects:

[0117] (1) The present invention provides a method for collecting intracellular nanovesicles derived from mesenchymal stem cells by ultrasonic cell disruption, which can avoid the cumbersome steps and impurities of collecting extracellular vesicles, that is, the operation of separating vesicles is simple, and the yield of the obtained vesicles is high. Compared with extracellular vesicles with exosomes as the main component, the obtained small intracellular nanovesicles have a smaller particle size, a narrow particle size distribution range, and are more stable at different temperatures. The obtained intracellular nanovesicles have good tissue compatibility. Compared with extracellular vesicles, the intracellular nanovesicles have a wider range and degree of distribution when injected in situ into tissues. When used as a carrier to load drugs, they have a higher encapsulation rate and drug loading rate. For example, the drugs loaded by intracellular nanovesicles in the form of intravitreal injection can be absorbed by the retina faster.

[0118] (2) The small intracellular nanovesicles prepared by the present invention have very good application and research value in nerve damage or neurodegenerative diseases (such as optic nerve damage, ischemic stroke, Alzheimer's disease, etc.). The small intracellular nanovesicles can inhibit the apoptosis of ganglion cells, slow down the reduction of GCC thickness, reduce neuronal pathological damage and the relative number of apoptotic cells, activate microglia and astrocytes, reduce inflammatory stress on neural tissue, increase the expression of β-tubulin Ⅲ, Occludin and ZO-1, improve the motor coordination ability and neuromuscular ability of animals, improve the learning disabilities and cognitive disabilities of animals, and stimulate the spontaneous exploratory behavior of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Figure 1 The figure shows a schematic diagram of the process and steps of the method for producing intracellular nanovesicles. Figure 1 A shows a flow chart, Figure 1 B shows the steps.

[0120] Figure 2 The figure shows the optimization process of the separation parameters of intracellular nanovesicles. Figure 2 A- Figure 2 B shows the protein yield (A) and vesicle yield (B) of sIVs obtained at different action times and an ultrasonic amplitude of 20%. At an ultrasonic amplitude of 20%, the vesicle yield dropped sharply when the action time was less than 10 seconds or greater than 20 seconds. Figure 2 C- Figure 2 D shows the protein yield (C) and vesicle yield (D) of sIVs obtained at different ultrasonic amplitudes at an ultrasonic exposure time of 15 s. When the ultrasonic amplitude was higher than 25% at an ultrasonic time of 15 s, the vesicle yield dropped sharply. Figure 2 E shows the transmission electron microscopy images of sIVs obtained under different ultrasonic amplitudes at an ultrasonic exposure time of 15 s, scale bar: 100 nm. Figure 2 F shows the transmission electron microscopy images of sIVs obtained at 20% ultrasonic amplitude and different action times, scale bar: 100 nm.

[0121] Figure 3 Shown are transmission electron micrographs of sEVs and sIVs of MSCs cells. Wide field scale bar: 200 nm; close-up scale bar: 200 nm.

[0122] Figure 4 Shown are the results of nanoparticle size analysis, which show the particle size distribution of MSCs cells and their sEVs and sIVs.

[0123] Figure 5 Shown are the statistical analysis results of the sizes of MSCs cells, sEVs and sIVs ( p<0.01, p<0.001 indicates significant difference among the groups).

[0124] Figure 6 Shown are the statistical analysis results of the number of sEVs and sIVs vesicles (A) and total protein production of MSCs cells under equal cell numbers (B) p<0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).

[0125] Figure 7 Shown are the results of Coomassie Brilliant Blue staining, which show the protein distribution of MSCs cells and their sEVs and sIVs.

[0126] Figure 8 Shown are Western blot results, which show the expression of exosome marker proteins (Alix, HSP70, TSG101, CD63, CD81) of MSCs cells.

[0127] Fig. 9 Shown are transmission electron microscopy images of sEVs and sIVs of MSCs at different temperatures. Scale bar: 200 nm.

[0128] Fig.10 The results of nanoparticle size analysis of sEVs and sIVs of MSCs cells at different temperatures (-80°C, 4°C and 37°C) are shown. Fig.10 A shows the particle size distribution, Fig.10 B shows the statistical analysis results of particle size ( p<0.05, p<0.01 indicates significant difference among the groups).

[0129] Fig.11 Shown are sIVs-specific proteins in MSCs cells, arranged from high to low abundance, showing the top 50 proteins with the highest expression.

[0130] Fig.12 The results are shown for super-resolution microscopy and total internal reflection fluorescence structured illumination microscopy. Fig.12 A shows the TIRF-SIM mode (showing the cell membrane), which shows that CD63-positive (green) areas exist on the cell membrane surface, while TMEM214-positive (red) areas are almost not observed. Fig.12 B shows the wide-field 2DSM mode (showing the whole cell), which demonstrates the simultaneous presence of CD63-positive and TMEM214-positive signals in the entire cell. Fig.12C shows time-phase screenshots of dynamic observation of living cells. The green on the left represents the late endosomes, sEVs and cell membrane marked by CD63. The arrow at 0s indicates that sEVs have just been released from the cell membrane to the outside of the cell, and gradually move away from the cell membrane from 6min to 14min. The red on the right represents TMEM214, indicating that sIVs are diffusely distributed in the cell and have not been released outside the cell.

[0131] Fig.13 Shown is a Venn diagram showing the total protein species in MSCs cells, sEVs, and sIVs.

[0132] Fig.14 Shown are the principal component analysis results of total proteins identified in MSCs cells, sEVs, and sIVs.

[0133] Fig.15 Shown is a heat map showing differentially expressed proteins between sEVs and sIVs of MSCs cells.

[0134] Fig.16 Shown is a volcano plot showing the top five significantly differentially expressed proteins between sEVs and sIVs of MSCs cells.

[0135] Fig.17 Shown is a heat map showing the differential expression of exosomal markers between sEVs and sIVs of MSCs cells.

[0136] Fig.18 Shown is a heat map showing the differential expression of organelle markers between sEVs and sIVs of MSCs cells.

[0137] Fig.19 Shown is a heat map showing the differential expression of Clathrin family proteins between sEVs and sIVs of MSCs cells.

[0138] Fig. 20 Shown is the cellular component enrichment analysis of sIVs-expressed proteins in MSCs, where “Summary” represents the representative pathway after item clustering analysis, and “Item” represents the pathway and the specific items of the pathway.

[0139] Fig.21 Shown is the biological process enrichment analysis of sIVs-expressed proteins in MSCs, where “Summary” represents the representative pathway after project cluster analysis, and “Project” represents the pathway and the specific items of the pathway.

[0140] Fig. 22 The figure shows the difference in cytokine levels of IL-1β and IGF2 carried by sEVs and sIVs of MSCs detected by protein profiling. Fig. 22A shows the difference in IL-1β cytokine levels carried by sEVs and sIVs of MSCs detected by protein profiling. Fig. 22 B shows the difference in cytokine levels of IGF2 carried by sEVs and sIVs of MSCs detected by protein profiling ( p<0.05, p<0.001 indicates significant difference among the groups).

[0141] Fig.23 The figure shows the difference in cytokine levels of IGF-1, EGF, IL-10, IL-6 and TNFα carried by sEVs and sIVs of MSCs detected by ELISA. Fig.23 A shows the difference in the cytokine levels of IGF-1 carried by sEVs and sIVs of MSCs detected by ELISA. Fig.23 B shows the difference in cytokine levels of EGF carried by sEVs and sIVs of MSCs detected by ELISA. Fig.23 C shows the difference in IL-10 cytokine levels carried by sEVs and sIVs of MSCs detected by ELISA. Fig.23 D shows the difference in IL-6 cytokine levels carried by sEVs and sIVs of MSCs detected by ELISA. Fig.23 E shows the difference in cytokine levels of TNFα carried by sEVs and sIVs of MSCs detected by ELISA ( p<0.01, p<0.001 indicates significant difference among the groups, ns indicates no statistical difference).

[0142] Fig.24 Shown are the relative RNA abundances in sEVs and sIVs of MSCs cells (ns indicates no statistical difference).

[0143] Fig.25 Shown are the percentages of small noncoding RNAs read from small RNAs in sEVs and sIVs from MSCs. miRNA: micro-RNA; snoRNA: small nucleolar RNA; snRNA: small nuclear RNA; tRNA: transfer RNA; rRNA: ribosomal RNA.

[0144] Fig.26 Shown is a Venn diagram showing the miRNA species contained in sEVs and sIVs of MSCs cells.

[0145] Fig. 27 Shown are the principal component analysis results of the MSCs cell miRNA dataset.

[0146] Fig.28Shown is a matchstick chart showing the top 10 highly abundant miRNAs in sEVs and sIVs of MSCs cells.

[0147] Fig.29 Shown is a heat map showing that there are more differentially expressed miRNAs between sEVs and sIVs of MSCs cells.

[0148] Fig.30 Shown is a volcano plot showing the top 5 differentially expressed miRNAs between sEVs and sIVs of MSCs cells. The horizontal axis represents the expression fold change (log2 difference fold) of miRNA between different samples or comparison combinations, and the vertical axis represents the significance level of the expression difference.

[0149] Fig.31 Enrichment analysis of candidate target genes of miRNAs differentially expressed in MSCs-derived sEVs and sIVs as shown. Fig.31 A shows the top 10 entries of biological process (BP), cellular component (CC) and molecular function (MF) in GO analysis. Fig.31 B shows the KEGG enrichment analysis of the differentially expressed miRNA candidate target genes in sEVs and sIVs of MSCs.

[0150] Fig.32 Shown are the types and proportions of metabolites contained in sEVs and sIVs of MSCs cells.

[0151] Fig.33 Shown are the principal component analysis results of lipids contained in MSCs cell sEVs and sIVs.

[0152] Fig.34 Shown is a heat map showing the differential lipid types contained in MSCs cell sEVs and sIVs.

[0153] Fig.35 The figure shows the lipidome bar graph of MSCs cell sIVs group versus sEVs group. The horizontal axis of the figure represents the relative change percentage of the content of each substance in the group. If the relative change percentage of the content is zero, it means that the content of the substance in the two groups is the same; the relative change percentage of the content is a positive number, indicating that the content of the substance in the sIVs group is higher; the relative change percentage of the content is a negative number, indicating that the content of the substance in the sEVs group is higher. The vertical axis of the lipidome bar graph represents the classification information of lipids.

[0154] Fig.36 The results show the ability of intracellular nanovesicles to be internalized by RPE cells cultured in vitro. Fig.36 A and B show that DiD-labeled sEVs and sIVs were co-incubated with RPE cells for 3h, 12h, 24h and 48h. The green color is the cytoskeleton, the red color is the vesicle, and the blue color is the cell nucleus shown by DAPI staining. Scale bar: 20μm. Fig.36 C shows the statistical results of DiD fluorescence intensity in cells ( p<0.05, p<0.01 and p<0.001 indicates significant difference among the groups).

[0155] Fig.37 The results show the ability of intracellular nanovesicles to be internalized by HRMECs cells cultured in vitro. Fig.37 A and B show that DiD-labeled sEVs and sIVs were co-incubated with HRMECs for 3h, 12h, 24h and 48h. The green color is the cytoskeleton, the red color is the vesicle, and the blue color is the cell nucleus shown by DAPI staining. Scale bar: 20μm. Fig.37 C shows the statistical results of DiD fluorescence intensity in cells ( p<0.05, p<0.01 and p<0.001 indicates significant difference among the groups).

[0156] Fig.38 The results show the ability of intracellular nanovesicles to be internalized by the retina. Fig.38 A shows the distribution of DiD-labeled sEVs and sIVs in retinal sections 24h and 48h after subconjunctival injection. Fig.38 B shows the distribution of DiD-labeled sEVs and sIVs in retinal sections 8h and 24h after intravitreal injection. DAPI staining shows cell nuclei. Scale bar: 20μm. Fig.38 C shows the statistical results of DiD fluorescence intensity in the retina of the subconjunctival injection group. Fig.38 D shows the statistical results of DiD fluorescence intensity in the retina of the intravitreal injection group ( p<0.05, p<0.01, p<0.001 and p<0.0001 indicates significant difference among the groups).

[0157] Fig.39 The results of the retinal flat-mount staining experiment after the ONC mouse model was successfully established are shown. It shows the changes in the number of retinal ganglion cells under different clamping times (A) and its statistical analysis results (B) ( p<0.0001 indicates significant difference among the groups).

[0158] Fig.40 The results of the retinal flat mount staining experiment of ONC mice at different treatment concentrations are shown. It shows the status of ONC mouse retinal ganglion cells (A) and its statistical analysis results (B), L represents low concentration (1.25mg / ml), H represents high concentration (2.5mg / ml) ( p<0.05, indicating significant difference among the groups).

[0159] Fig.41 The results of retinal flat mount staining experiments after treatment of ONC mice at different times are shown. It shows the status of ONC mouse retinal ganglion cells (A) and statistical analysis results after 7 days and 21 days of administration (B and C) ( p<0.05, indicating significant difference among the groups).

[0160] Fig.42 The results of optical coherence tomography experiments are shown. They show the structural changes of each layer of the retina at the optic disc level (A) and the statistical analysis results of the thickness of the retinal ganglion cell complex (B) ( p<0.05, p<0.01, p<0.001 indicates significant difference among the groups).

[0161] Fig.43 The results of the rotarod test in each group of stroke mice are shown. It shows that the time for the mice in the PBS group to fall from the rotarod test was significantly earlier than that in the MSC-sEVs group and the MSC-sIVs group, and the time was shorter. The time for the experimental animals in the MSC-sEVs group and the MSC-sIVs group to fall from the rotarod was higher than that in the PBS group on the 3rd, 5th and 7th days.

[0162] Fig.44 The results of the forelimb gripping test of each group of stroke mice are shown. It shows that ischemia induced a decrease in forelimb gripping strength at various time points within 7 days after ischemia, while administration of MSC-sIVs and MSC-sEVs significantly increased forelimb gripping strength 3 days after ischemia.

[0163] Fig.45 The results of TTC tissue staining of a mouse model of stroke are shown. It shows that after TTC staining, the white infarct area in the PBS group was the largest, while the MSC-sIVs and MSC-sEVs groups showed less white infarct area, and there were significant differences between the PBS group and MSC-sEVs and MSC-sIVs.

[0164] Fig.46The results of TUNEL assay for apoptosis of neurons in the brain tissue of mice with stroke in each group are shown. Compared with the PBS group, the MSC-sIVs and MSC-sEVs treatment groups significantly reduced the number of apoptotic neurons in the cerebral cortex of mice with photochemical stroke, and the difference was statistically significant compared with the PBS group ( p<0.05).

[0165] Fig.47 The results are shown for staining of IBa-1, a specific marker of microglia, in the brain tissues of stroke mice in each group. The activation of microglia in the cortical ischemic area in the MSC-sIVs and MSC-sEVs groups was reduced compared with that in the PBS group ( p<0.05 indicates significant difference between the groups). Compared with MSC-sEVs, the intervention of MSC-sIVs significantly reduced the number of activated microglia.

[0166] Fig.48 The results of immunofluorescence of GFAP, a marker of astrocyte activation, are shown. The results show that the expression of GFAP protein in the brain tissue of stroke mice in the PBS group was significantly higher than that in the MSC-sIVs group and the MSC-sEVs group. MSC-sIVs can reduce GFAP expression and the effect is more significant than that of MSC-sEVs ( p<0.05, p<0.001 and p<0.0001 indicates significant difference among the groups).

[0167] Fig.49 The results of immunofluorescence staining of β-tubulin Ⅲ protein in the brain tissue of stroke mice are shown. It shows that MSC-sEVs treatment can significantly increase the expression level of β-tubulin Ⅲ protein in the brain tissue of mice ( p<0.05 indicates significant difference among the groups), and MSC-sIVs can increase the expression of β-tubulin Ⅲ and the effect is more significant than MSC-sEVs.

[0168] Fig.50 The results of immunofluorescence staining of CD 31, Occludin and ZO-1 in brain tissue of stroke mice are shown. It shows that the expression of CD 31, Occludin and ZO-1 in brain tissue of mice in PBS group was significantly decreased, and compared with PBS group, the levels of CD 31, Occludin and ZO-1 in brain tissue were significantly increased after MSC-sIVs and MSC-sEVs treatment.

[0169] Fig.51 Shown are the results of the Morris water maze experiment on Alzheimer's disease mice. Fig.51A shows that in the navigation experiment (finding the platform), the escape latency of the PBS group mice from the 4th to the 6th day was significantly higher than that of the normal group, while the escape latency of the 5xFAD mice treated with MSC-sIVs and MSC-sEVs was significantly lower than that of the 5xFAD mice treated with PBS on the 4th to 6th day. In addition, the latency of the MSC-sIVs group to reach the platform was significantly shorter than that of the PBS group on the 4th and 6th day of navigation, and the difference was statistically significant ( p<0.05). Fig.51 B shows the positioning navigation stage on the 6th day. We found that PBS mice had difficulty finding the platform and swam around the edge compared with the normal, MSC-sIVs, and MSC-sEVs groups, indicating that the mice in the PBS group had more severe spatial memory impairment.

[0170] Fig.52 The results of the open field test on Alzheimer's mice are shown. It shows that the activity trajectories of mice in the PBS group tend to be in the peripheral area of ​​the open field.

[0171] Fig.53 The results of the Y-maze experiment on Alzheimer's mice are shown. It shows that the spontaneous alternation rate of mice in the PBS group was significantly reduced; compared with the PBS group mice, the spontaneous alternation rates of mice in the normal group, MSC-sIVs and MSC-sEVs were significantly increased ( p<0.05, p<0.01 indicates significant difference between groups), indicating that the learning and memory dysfunction of these mice was alleviated after treatment. DETAILED DESCRIPTION

[0172] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0173] The term "patient" or "subject" and the like are used interchangeably herein and refer to any animal or cell thereof treated according to the methods described herein, whether in vitro or in situ. Specifically, the aforementioned animals include mammals, e.g., rats, mice, guinea pigs, rabbits, dogs, monkeys, humans, particularly humans.

[0174] The term "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, inhibiting, suppressing and / or halting one or more clinical symptoms of a disease after onset of the disease.

[0175] The term "prevent" refers to avoiding, minimizing or making the onset or development of a disease difficult by treating it before it occurs.

[0176] The term "intracellular vesicle" is also called intracellular nanovesicle, small intracellular vesicle, and small intracellular nanovesicle.

[0177] The term "extracellular vesicles" is also called small extracellular vesicles, which are a type of vesicles with a double membrane structure that are secreted by cells into the extracellular environment.

[0178] The nerve damage in the present invention may be caused by various reasons, for example, infection (some viruses, bacteria, fungi, parasites and other pathogens can cause nerve damage), trauma (such as chemical damage, photochemical damage, knife cuts, burns, firearm injuries, abrasions, extrusions, impacts and other nerve damage), compression or traction injuries (such as common clinical intervertebral disc herniation or spinal canal stenosis, resulting in compression of spinal nerves and spinal cord, brachial plexus injury and lower limb nerve damage caused by women during pregnancy and childbirth), autoimmune mechanism (such as myasthenia gravis, multiple sclerosis and other nerve damage caused by), ischemic injury (such as stroke, cerebral thrombosis, etc.), ischemic nerve damage caused by formation), metabolic diseases (such as nerve damage caused by peripheral neuropathy such as diabetes), tumors, hereditary, nutritional, inflammatory, and drug damage (such as nerve damage caused by isoniazid and metformin affecting the absorption of vitamin B12). Its manifestations may include, but are not limited to, movement disorders (such as muscle weakness, paralysis or tremor), sensory disorders (such as numbness, tingling, paresthesia or loss of sensation), autonomic nervous system disorders (such as hypotension, syncope, constipation or urinary incontinence), cognitive disorders (such as amnesia, confusion or inattention), and mental disorders (such as depression, anxiety, abnormal behavior or mental confusion).

[0179] The term "neurological injury-related diseases" is also called neurological injury diseases, which refers to diseases that affect the functions of the nervous system, including but not limited to spinal cord diseases, optic nerve diseases, cerebrovascular diseases, traumatic brain injury diseases, extrapyramidal diseases (also known as movement disorders), demyelinating diseases, neurodegenerative diseases, central nervous system infectious diseases, cerebral palsy, hydrocephalus, cranial nerve diseases, nerve root diseases, plexus diseases, single nerve diseases, multiple nerve diseases, neuromuscular junction diseases, ataxia, leukodystrophy, mental retardation, familial amyloid polyneuropathy (FAP), Charcot-Marie-Tooth disease (CMT, also known as hereditary motor sensory neuropathy (HMSN)), neurosis (such as hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, somatoform disorder).

[0180] The term "neurodegeneration-related diseases" is also called neurodegenerative diseases or nervous system degenerative diseases. It is a group of diseases with unknown causes that chronically progressively damage the central nervous system and peripheral nervous system and other tissues, including but not limited to motor neuron disease, Alzheimer's disease, multiple system atrophy (MSA), senile cerebral atrophy, Pick's disease dementia, alcoholic nervous system degeneration, frontotemporal dementia, subacute necrotizing encephalopathy (also known as Leigh syndrome), corticostriatal degeneration (CJD, also known as Creutzfeldt-Jakob disease), dementia with Lewy bodies (DLB), Machado-Joseph disease (MJD, also known as Azores disease).

[0181] The term "spinal cord disease" refers to diseases caused by injury or damage to the spinal cord, including but not limited to spinal cord injury, myelitis, poliomyelitis, encephalomyelitis, spinal cord tumors, spinal muscular atrophy (SMA), motor neuron disease, spinal cord compression, syringomyelia, syringomyelia, vascular myelopathy (such as spinal cord hemorrhage, spinal cord ischemia, spinal cord embolism, spinal cord infarction, spinal cord necrosis, spinal cord edema, spinal artery thrombosis, posterior spinal artery syndrome, anterior spinal artery occlusive syndrome, spinal vascular malformations, spinal arteriovenous malformations (AVMs)), hepatic myelopathy, cervical spondylotic myelopathy, spinal arachnoiditis, epidural abscess, spondylosis, cauda equina syndrome, HTLV-1 associated myelopathy / tropical spastic paralysis (HAM / TSP).

[0182] The term "optic nerve disease" refers to diseases that occur in the optic nerve. Any interruption of the function of the optic nerve due to any cause (vascular, compressive, toxic or developmental abnormalities) will cause optic nerve diseases, which have the common feature of visual impairment, including but not limited to optic nerve tumors, ischemic optic neuropathy, hereditary optic neuropathy, compressive optic neuropathy, optic nerve injury (also known as traumatic optic neuritis, traumatic optic neuropathy, traumatic optic nerve injury, TON), nutritional optic neuropathy, toxic optic neuropathy, optic neuritis, papilledema, optic nerve atrophy, and glaucoma.

[0183] The term "cerebrovascular disease" refers to a group of diseases that occur in the blood vessels of the brain and cause brain tissue damage due to impaired intracranial blood circulation, including but not limited to ischemic cerebrovascular disease (such as cerebral ischemia, cerebral infarction (also known as ischemic stroke), cerebral thrombosis, cerebral embolism, ischemic-hypoxic encephalopathy), hemorrhagic cerebrovascular disease (such as cerebral hemorrhage, subarachnoid hemorrhage, non-traumatic subdural hemorrhage, non-traumatic epidural hemorrhage), cerebral atherosclerosis, cerebral arteritis, cerebral aneurysm, cerebral arteriovenous fistula, amyloid cerebrovascular disease, intracranial venous thrombosis, intracranial blood pressure malformation (such as cerebral vascular arteriovenous malformation, cerebral cavernous malformation, dural arteriovenous fistula, carotid cavernous sinus fistula), intracranial telangiectasia, reversible cerebral vasoconstriction syndrome, vascular dementia (such as Binswanger's disease, multiple infarct dementia, hemorrhagic dementia), hypertensive encephalopathy, moyamoya disease, red core syndrome, Weber syndrome, Benedict's syndrome, and vascular leukoencephalopathy.

[0184] The term "extrapyramidal disease" refers to a group of unique movement disorders caused by extrapyramidal pathological changes mainly in the basal ganglia of the brain, mainly manifested by dysfunction of voluntary movement regulation, muscle strength, sensation and cerebellum are not affected, including but not limited to Hallevorden-Spatz disease (HSD, also known as globus pallidus nigra and red degeneration), progressive supranuclear palsy, multiple system atrophy, calcification of the basal ganglia (Fahr's disease), dystonia, orofacial dyskinesia, Meige syndrome, essential tremor, myoclonus, chorea, restless legs syndrome, stiff-person syndrome, and Wilson's disease.

[0185] The term "demyelinating disease" refers to an autoimmune system disease characterized by multifocal and inflammatory demyelination of the central nervous system, including but not limited to, multiple sclerosis (such as brainstem multiple sclerosis, spinal cord multiple sclerosis, multiple sclerosis dementia), acute disseminated encephalomyelitis (ADEM), neuromyelitis optica, acute hemorrhagic leukoencephalitis (AHLE), diffuse sclerosis, central pontine myelinolysis, extrapontine myelinolysis, acute transverse myelitis, subacute necrotizing myelitis (FAS), concentric sclerosis (Balo disease), tumor-like demyelinating lesions (TDLs), cerebral white matter demyelination, demyelinating myelitis, and clinically isolated syndrome.

[0186] The term "neurodegenerative disease" refers to a disorder caused by the loss of neurons and / or their myelin sheaths that worsens over time and results in functional impairment, including but not limited to epilepsy, cerebral ischemia (CI), brain injury (BI), Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), ataxia-telangiectasia syndrome, bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, multiple sclerosis (MS), primary lateral sclerosis (PLS), spinal muscular atrophy, and spastic paraplegia.

[0187] The term "infectious diseases of the central nervous system" includes diseases caused by viruses, bacteria, fungi, rickettsia, spirochetes, parasites, etc., including but not limited to encephalitis, meningitis (such as pachymeningitis, leptomeningitis, arachnoiditis), myelitis, encephalomyelitis, ventriculitis, cerebellitis, brainstemitis, intracranial abscess (such as brain abscess, epidural abscess, subdural abscess), intracranial granuloma (such as brain granuloma, epidural granuloma, subdural granuloma), neurosyphilis, brain parasitic disease, and prion disease.

[0188] The term "cerebral palsy", also known as cerebral palsy, is a syndrome caused by non-progressive brain damage and developmental defects from conception to infancy, mainly manifested by movement disorders and abnormal postures, including but not limited to spastic cerebral palsy, dyskinetic cerebral palsy, ataxic cerebral palsy, and Worster-Drought syndrome.

[0189] The term "hydrocephalus" refers to a buildup of fluid in the deep cavities of the brain that causes pressure and functional problems on the brain, including but not limited to, communicating hydrocephalus, obstructive hydrocephalus, compensated hydrocephalus, and congenital hydrocephalus.

[0190] The term "cranial nerve disease" refers to diseases caused by damage or dysfunction of the areas of the brain that control cranial nerves, nerve fibers connecting cranial nerve centers in the brain, or cranial nerves, including but not limited to trigeminal nerve diseases (such as trigeminal neuralgia), facial nerve diseases (such as Bell's palsy, geniculate ganglionitis (also known as Hunter's syndrome, Ramsay-Hunt syndrome), Mero syndrome, hemifacial spasm, hemifacial atrophy, facial neuritis), olfactory nerve diseases, glossopharyngeal nerve diseases, vagus nerve diseases, hypoglossal nerve diseases, and multiple cranial nerve diseases.

[0191] The term "radiculopathy" refers to a class of acute or chronic diseases caused by long-term compression of nerve roots by lesions in or adjacent to the spine, including but not limited to nerve compression and nerve root sleeve cysts.

[0192] The term "plexus disease" refers to diseases of the brachial plexus or lumbosacral plexus causing mixed motor and sensory disturbances, occurring in the upper or lower extremities, respectively, including but not limited to brachial plexus injury and lumbosacral plexus injury.

[0193] The term "mononeuropathy" refers to damage to a single peripheral nerve and includes, but is not limited to, upper extremity mononeuropathies (e.g., carpal tunnel syndrome, ulnar neuropathy, radial nerve damage) and lower extremity mononeuropathies (e.g., sciatic nerve damage, femoral neuropathy, tibial neuropathy, tarsal tunnel syndrome, plantar nerve damage, intercostal neuropathies).

[0194] The term "polyneuropathy" refers to a group of diseases caused by diffuse peripheral nerve disorders whose symptoms are not limited to a single nerve distribution area or a single limb and are often bilaterally symmetrical, including but not limited to idiopathic progressive neuropathies, inflammatory polyneuropathy, and secondary polyneuropathy (such as diabetic polyneuropathy, infectious polyneuropathy, and nutritional polyneuropathy).

[0195] The term "neuromuscular junction disease" refers to a group of diseases with dysfunctional transmission at the neuromuscular junction, including but not limited to myasthenia gravis, myasthenic syndrome, muscular dystrophy, myotonia-related diseases (such as neuromyotonia, myotonia atrophica, dystrophic myotonia, myotonia congenita, paramyotonia), mitochondrial myopathy, alcoholic myopathy, inflammatory myopathy, muscular atrophy, periodic paralysis (also known as periodic paralysis), ocular myopathy, and myofibrillar myopathy.

[0196] The term "ataxia" refers to a disorder of coordinated movement with normal muscle strength, including, but not limited to, cerebellar ataxias (such as Marinesco-Sjogren syndrome, Ramsay-Hunt syndrome (also known as Hunter syndrome), episodic ataxia (EA), Friedreich ataxia (FA), spinocerebellar ataxia (SCA), hereditary spastic ataxia (also known as Marie ataxia), Kearn-Sayre syndrome (KSS), ataxia telangiectasia (also known as Louis Bar syndrome), CANVAS syndrome), hereditary spastic paraplegia (HSP, also known as Strumpell-Lorrain disease).

[0197] The term "neurosis", also known as neurosis, is a group of mental disorders characterized by decreased mental activity, worry, tension, anxiety, depression, fear, compulsion, hypochondriasis, dissociative symptoms, conversion symptoms or neurasthenia, including but not limited to hysteria, anxiety disorder, obsessive-compulsive disorder, autism, depression, panic disorder, neurasthenia, phobia, hypochondriasis, and somatoform disorder.

[0198] The term "motor neuron disease" refers to a group of chronic progressive degenerative diseases that mainly damage the anterior horn of the spinal cord, the motor nuclei of the pontine and medullary cranial nerves, and the pyramidal tracts, including but not limited to amyotrophic lateral sclerosis (ALS), progressive spinal muscular atrophy (PMA), pyramidal tract degeneration, progressive bulbar palsy (PBP), pseudobulbar palsy, true bulbar palsy, primary lateral sclerosis (PLS), spinal bulbar muscular atrophy (SBMA, also known as Kennedy's disease), lower motor neuron syndrome, upper motor neuron syndrome, flail arm syndrome, and Madras motor neuron disease.

[0199] In the examples of the present invention, each experiment was repeated 3 times or more, and statistical analysis was performed using GraphPad Prism 90 software, and the data were expressed as mean ± standard error. One-way analysis of variance was used for comparison of data between two or more groups, and the differences between the groups were detected by Bonferroni post hoc test and nonparametric Kruskal-Wallis test with Dunn's multiple comparison test, and p < 0.05 was considered statistically significant.

[0200]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0201] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0202] Example 1: Extraction and culture of mesenchymal stem cells (MSC)

[0203] Human umbilical cords were provided by Beijing Beilai Biotechnology Co., Ltd., China. Umbilical cords obtained from normal pregnancies without complications after cesarean section were immediately placed in saline containing penicillin (100 U / mL) and streptomycin (100 mg / mL) and then transported to the laboratory within 4 hours. After removing residual blood and blood vessels, the obtained umbilical cords were cut into 1-3 mm pieces and digested with 0.1% type II collagenase for 1 hour at 37°C. The suspension was then filtered through a 100-mesh screen to remove undigested tissue. The supernatant from the filtration was centrifuged and washed three times with PBS. The cell pellet was resuspended in Dulbecco's modified Eagle's medium / nutrient mixture F12 complete medium. The medium contained 10% fetal bovine serum (FBS), 100 U / ml penicillin, and 100 mg / ml streptomycin. The cells were seeded in T175 culture flasks and cultured in a 5% CO2 incubator at 37°C. The medium was changed every 3 days. When the cell confluence reached 80%, the cells were passaged at a subculture ratio of 1:2, and cells from P3 to P5 were used for experiments.

[0204] At the same time, the above cell culture can refer to the Chinese patent application with publication number CN118207158A (publication date: 2024.06.18).

[0205] Example 2: Preparation of cell-derived nanovesicles

[0206] The production process of intracellular nanovesicles is as follows Figure 1 shown.

[0207] 1. Cell digestion and counting

[0208] When the cells grow to 90% confluence, the cell supernatant is aspirated, the cells are washed twice with PBS, and the cells are digested with trypsin. After digestion, the cells are neutralized and washed three times with PBS. Then the cells are counted and the cell number is adjusted to 1×10 with PBS. 6 Pieces / mL.

[0209] 2. Ultrasonic treatment of cells

[0210] Take 1×10 6 2 mL of cell suspension with a density of cells / mL was added to the bottom of a 50 mL centrifuge tube, and the ultrasonic probe was placed in the center of the liquid surface. The ultrasonic amplitude parameters were 20%, the time parameters were 15s, on 2s, off 2s, and the centrifuge tube was placed on ice. The liquid was then transferred to a 2 mL centrifuge tube for centrifugation.

[0211] 3. Collection of Nanovesicles in Small Cells

[0212] The centrifugation parameters were 2000g×10min, 20000g×30min. The supernatant was then collected and transferred to an ultracentrifuge tube, and the centrifugation parameters were 150000g×70min. All the above operations were performed on ice. The obtained precipitate was resuspended in PBS and was small intracellular nanovesicles (sIVs).

[0213] 4. Collection of small extracellular vesicles

[0214] FBS is a necessary condition for in vitro cell culture, but it contains a large number of bovine extracellular vesicles, which will also be present in the complete cell culture medium containing FBS. In order to remove bovine extracellular vesicles, FBS was centrifuged at 110,000g overnight (about 12 hours) at 4°C. When the cell fusion reached 60%, the cells were cultured in complete culture medium containing 10% FBS with exosomes removed for 48 hours. Then, the supernatant was collected and the extracellular vesicles were separated by ultracentrifugation at 4°C. The specific steps included 300g×10min, 2000g×10min, 10000g×30min and 110000g×70min twice. The obtained precipitate was resuspended in PBS, which was small extracellular vesicles (sEVs) mainly composed of exosomes.

[0215] Example 3: Optimization of isolation parameters for intracellular nanovesicles

[0216] Referring to the steps of Example 2, the optimization process of the separation parameters of intracellular nanovesicles is as follows: Figure 2 shown.

[0217] 1. Nanoparticle size analyzer detects the difference in the yield of sIVs

[0218] Take the PBS-resuspended sIVs, dilute them to 1 ml with PBS, and use the Nanoparticle Tracking Analysis (NTA) software NTA 3.3 Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the Mode particle size result. The camera mode is sCMOS. The laser type is Blue488, and the viscosity is 0.9 cP.

[0219] 2. Observation of the morphology of sIVs in different cells by transmission electron microscopy

[0220] Take the centrifuged sIVs, resuspend them in 200μL PBS solution and mix them evenly. Take 10μL sIVs solution and mix it with 4% PFA in a volume ratio of 1:1, drop it on a clean plastic film to form droplets, then put the front of the electron microscope carbon grid on the droplets and leave it for 20 minutes. Negatively stain with 10μL phosphotungstic acid for 90s, bake the carbon grid dry, and observe it using a HitacW-7500 transmission electron microscope.

[0221] Figure 2 A and Figure 2 B shows the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasonic amplitude of 20% and an action time of 5s, 10s, 15s, 20s, 25s, 30s and 60s. The results show that when the action time is less than 10 seconds or more than 20 seconds, the number of vesicles and protein yield drop sharply. Figure 2 C and Figure 2 D shows the protein yield and vesicle yield of sIVs obtained according to the implementation steps at an ultrasound time of 15 s and an amplitude of 20%, 25%, 30%, 35% and 40%, respectively. The results show that at an ultrasound time of 15 s, when the ultrasound amplitude is higher than 25%, the vesicle yield drops sharply. Figure 2 E shows the transmission electron microscopy images of sIVs obtained according to the present implementation steps at an ultrasonic time of 15 s and ultrasonic amplitudes of 20%, 25%, 30%, 35% and 40%. Figure 2 F shows the transmission electron microscopy images of sIVs obtained according to the implementation steps at an ultrasonic amplitude of 20% and ultrasonic times of 5s, 10s, 15s, 20s, 25s, 30s and 60s.

[0222] This optimization process shows that at an ultrasonic amplitude of 20%, the vesicle yield drops sharply when the action time exceeds 10 seconds or 20 seconds. At an ultrasonic time of 15 seconds, the vesicle yield drops sharply when the ultrasonic amplitude is higher than 25%. 20% amplitude and 15 seconds ultrasonic time are the best parameters for collecting intracellular nanovesicles.

[0223] Regarding the preparation of cell vesicles, reference may be made simultaneously to the Chinese patent application with publication number CN118207158A (publication date: 2024.06.18).

[0224] Example 4: sIVs have unique physical characteristics and high thermal stability

[0225] 1. Experimental instruments and materials

[0226] 1.1 Experimental Reagents

[0227] Table 1 Experimental reagents

[0228]

[0229] 1.2 Experimental instruments

[0230] Table 2 Experimental instruments

[0231]

[0232] 2. Experimental methods

[0233] 2.1 Transmission electron microscopy observation of the morphology of sIVs and sIVs

[0234] Take the centrifuged sEVs and sIVs (prepared in Example 2), resuspend them in 200 μL PBS solution and mix them evenly, take 10 μL of sEVs and sIVs solution and mix them with 4% PFA in a volume ratio of 1:1, drop them on a clean plastic film to form droplets, then put the front side of the carbon mesh on the droplets and leave it for 20 minutes, negatively stain with 10 μL phosphotungstic acid for 90 seconds, bake the carbon mesh dry, and observe it using a HitacW-7500 transmission electron microscope.

[0235] 2.2 Nanoparticle size analysis to detect the particle diameter of sEVs and sIVs

[0236] Take the sEVs and sIVs resuspended in PBS, dilute them to 1 ml with PBS, and use Nanoparticle Tracking Analysis (NTA) 3.3 Dev Build 3.3.104 for detection. Set the temperature to 25°C, the laser to Blue 488, the flow rate to 50, the mode to automatic detection, inject three times, analyze three times, and take the peak value average as the particle size result of Mode. The camera mode is set to sCMOS. The laser type is set to Blue488, and the viscosity is set to 0.9 cP.

[0237] 2.3 Analysis of protein composition of sEVs and sIVs by Coomassie Brilliant Blue staining

[0238] After BCA quantification, take an equal amount of protein sample, add PBS to 20 μl, add 5 μl protein loading buffer (5×), and heat at 95°C for 5 minutes. The protein electrophoresis conditions are 100V, 90min. After electrophoresis, add Coomassie Brilliant Blue Ultrafast Staining Solution, incubate at room temperature for 2 hours, and wash with pure water until the water becomes clear. Photograph the gel.

[0239] 2.4 Western Blot analysis of exosomal marker protein composition of sIVs and sEVs

[0240] After protein detection by BCA kit, take equal amounts of protein samples from each group, add PBS to equal volume, add protein loading buffer (5×), and heat at 95℃ for five minutes. Prepare gel according to the instructions of SDS-PAGE kit and insert electrophoresis comb. Let it stand and wait for solidification at room temperature for 25 minutes. Place the prepared SDS gel in the pre-prepared electrophoresis tank. Remove the electrophoresis comb and inject the denatured protein into the SDS-PAGE loading tank. Add 1-4 μl of marker on each side. Adjust the voltage to 60V. After the upper gel runs, change the voltage to 100V and stop electrophoresis until the lowest bromophenol blue indicator line is 1-2cm away from the bottom of the glass plate. Place the activated PVDF membrane on the SDS-PAGE surface, and then place filter paper and sponge pads on both sides of the gel and membrane. Use the column to roll gently to remove bubbles in the system and clamp the electrotransfer clamp. Set the black electrode as SDS-PAGE and the red electrode as PVDF membrane, and transfer the membrane under constant pressure, while placing ice bags in the electrotransfer tank to cool down. After electrotransfer, place the PVDF membrane in a TBST solution containing 5% skim milk or 1% BSA and block it at room temperature for 2 hours. After blocking, add the primary antibody and incubate overnight on a shaker in a 4°C refrigerator. The next day, remove the primary antibody and add TBST solution to wash the PVDF membrane. Then, add the secondary antibody and incubate on a shaker at room temperature for 2 hours. After incubation, remove the secondary antibody again and add TBST solution to wash the PVDF membrane 3 times, 10 minutes each time. Finally, use ECL supersensitive luminescent liquid to develop the PVDF membrane.

[0241] 3. Statistical processing

[0242] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. SPSS22.0 was used to analyze all quantitative data. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. Nonparametric tests were used for non-normally distributed data and data with unequal variance. P Values ​​< 0.05 were considered statistically significant.

[0243] 4. Experimental results

[0244] 4.1 Transmission electron microscopy reveals the morphology of sEVs and sIVs

[0245] The sIVs of MSCs were enriched and detected using transmission electron microscopy. Figure 3 As shown in the figure, sEVs of MSCs cells are round or horseshoe-shaped with a diameter of 100-200nm, while sIVs are more numerous, round in shape, and less than 100nm in diameter. Electron microscopy results show that the diameter of sIVs is significantly smaller than that of sEVs.

[0246] 4.2 Nanoparticle tracking analysis reveals the size distribution of sEVs and sIVs

[0247] Nanoparticle size test results are as follows Figure 4 As shown, the particle size distribution range of sEVs from MSCs cells is wide and the particle size is larger, while the particle size distribution range of sIVs is narrow and the particle size is smaller.

[0248] After statistical analysis, the results are as follows Figure 5 As shown, the average particle size of sEVs of MSC is 123.1±4.453nm, and the average particle size of sIVs is 75.28±9.067nm; the particle size of sIVs is smaller than that of sEVs.

[0249] 4.3 Comparison of the yields of sEVs and sIVs at equal cell weights

[0250] To compare the yields of the two types of vesicles, we collected sEVs and sIVs from cell culture supernatants and adherent cells at the same time. 7 The number of sIVs produced by cells is 10- to 20-fold higher than that of sEVs ( Figure 6 A), derived from 1×10 7 The protein production in cellular sIVs is 20- to 40-fold higher than that in sEVs ( Figure 6 B). This indicates that the production of sIVs is much higher than that of sEVs.

[0251] 4.4 Differences in protein distribution between sEVs and sIVs

[0252] The whole proteins of cells, sEVs and sIVs were separated by SDS-PAGE, and the total protein distribution was displayed by Coomassie Brilliant Blue staining. The staining results showed that the proteins contained in cells showed the most abundant bands, with multiple high-abundance protein bands; sEVs contained fewer types of proteins, and the high-abundance proteins were located around 200kD and 70kD; sIVs contained more types of proteins than sEVs, and the high-abundance proteins were located around 250kD and 55kD ( Figure 7 The protein distributions varied between different cells, which preliminarily suggests that sEVs and sIVs have different protein compositions and are different from the total protein distributions of both cells and sEVs.

[0253] To further analyze the protein expression characteristics of cells and their sEVs and sIVs, Western blot was used to detect the expression of exosome marker proteins Alix, HSP70, TSG101, CD63, and CD81 in cells and their sEVs and sIVs under equal protein conditions. The results are shown in Figure 8As shown in the figure. The sEVs of cells express the most exosome marker proteins, and the cells themselves also express a certain amount of Alix, HSP70, TSG101, and CD63; however, Alix and CD81 of sIVs are basically not expressed, and the expression levels of HSP70, TSG101 and CD63 are also much lower than those of cells and sEVs, which further indicates that sIVs do not have the characteristics of exosomes and are not the precursors of exosomes in cells.

[0254] 4.5 Comparison of the stability of sEVs and sIVs at different temperatures

[0255] To evaluate the stability of both vesicles at different temperatures, the sIVs and sEVs suspensions were equally divided into three parts and stored at different temperatures (-80°C, 4°C, and 37°C). After 24 h, the morphology, size, and amount of protein of sEVs and sIVs were evaluated. Both vesicles were stable at -80°C and 4°C. However, TEM images showed that the morphology of sEVs was impaired at 37°C, with irregular shapes, broken vesicles, and rough borders ( Fig. 9 ), and the number of sEVs also decreased ( Fig.10 B), while the morphology and particle number of sIVs remained stable at 37°C. The above results indicate that sIVs have higher thermal stability than sEVs.

[0256] 4.6 Super-resolution imaging shows that the distribution of sIVs in cells is different from that of sEVs

[0257] To gain a deeper understanding of the intracellular distribution of sIVs, we used proteomic analysis to identify proteins uniquely expressed in sIVs compared to sEVs, namely IV signature proteins. We made the signature proteins carry green fluorescent protein and performed intracellular imaging to visualize the morphology of sIVs in cells. By performing proteomic analysis on sIVs and sEVs, we identified proteins uniquely expressed in sIVs. The protein expression abundance was ranked from high to low, and the top 50 proteins were displayed ( Fig.11). We observed that the expression abundance of TMEM214 protein was the highest in MSCs. TMEM214 is a transmembrane protein involved in cellular processes such as vesicle transport and protein transport (Zhao J., Xu J., Wang Y., et al. Membrane LocalizedGbTMEM214s Participate in Modulating Cotton Resistance to Verticillium Wilt.Plants (Basel). 2022 Sep 8;11(18):2342.). Therefore, we used green fluorescent protein GFP to mark TMEM214 to visually display the status of sIVs in cells, and used GFP-labeled CD63 as a marker for sEVs in cells.

[0258] Super-resolution microscopy combined with total internal reflection fluorescence microscopy (TIRF-SIM) showed that CD63 was located on the cell membrane ( Fig.12 A, green), TMEM214 is not expressed on the cell membrane ( Fig.12 A, red). This finding excludes the possibility that sIVs originate from cell membrane remodeling and confirms that the origin and activity site of sIVs are located within the cell. Super-resolution microscopy using Wildfield-2DSM scanning mode provides an overview of protein expression throughout the cell, and the image shows that both CD63 and TMEM214 are significantly expressed in the cell ( Fig.12 B). Subsequently, we observed the dynamic changes of these protein marker structures in living cells under wide field conditions. We took pictures every 10 seconds for 15 minutes to form a dynamic video. In the video screenshot, we can observe the dynamic release of sEVs from the cell membrane to the outside of the cell ( Fig.12 C, green, white arrows), while TMEM214-labeled sIVs were diffusely distributed in the cells and were not released outside the cells ( Fig.12 C, red). Intuitive microscopic imaging shows that sIVs are diffusely distributed in the cell in a cloud-like manner and are not released outside the cell.

[0259] 5. Summary

[0260] In this example, we took MSCs cells as an example to collect and characterize sIVs and sEVs. Through transmission electron microscopy and nanoparticle size analysis, it was found that the size of sIVs was significantly smaller than that of sEVs; the total protein expression patterns of cells, sEVs and sIVs were different, and sIVs expressed low exosome marker proteins; under the same number of cells, the yield of sIVs was significantly more than that of sEVs. Under -80°C conditions, the stability of sIVs and sEVs was comparable, but under 37°C conditions, the stability of sIVs was significantly better than that of sEVs. Super-resolution imaging was used to observe that sEVs were released to the outside of the cell through the cell membrane, while sIVs were frequently active inside the cell. In general, the sIVs vesicles collected by the method described in the present invention contain a unique protein composition, are highly stable at physiological temperature, and have a yield much higher than that of extracellular vesicles.

[0261] Example 5: Quantitative proteomic analysis shows that sIVs have unique protein expression profiles

[0262] 1. Experimental instruments and materials

[0263] 1.1 Experimental Reagents

[0264] Table 3 Experimental reagents

[0265]

[0266] 1.2 Experimental instruments

[0267] Table 4 Experimental instruments

[0268]

[0269] 2. Experimental methods

[0270] 2.1 Sample preparation for protein profiling of cells and their sEVs and sLVs

[0271] 2.1.1 Protein extraction

[0272] 1) Add 220ul of urea lysis buffer (8M urea, 50mM NH4HCO3, protease inhibitors) to cells, sEVs, and sIVs (prepared in Example 2) respectively, and lyse at room temperature for 5 minutes.

[0273] 2) Ultrasonic disruption on ice (energy 35%, ON 3S, OFF 3S, total ultrasonic time 2min), insert the sample tube into the ice box, set the centrifuge temperature to 20℃, centrifuge at 14,000g for 10min, take the supernatant, and repeat the centrifugation once.

[0274] 3) The protein concentration was detected by BCA method, and 100 μg protein was taken from each cell, sEVs, and sIVs.

[0275] 2.1.2 Protein denaturation

[0276] 1) Add DTT to each sample tube of cells, sEVs, and sIVs to a final concentration of 10 mM and incubate at 37°C for 1 hour to reduce the proteins.

[0277] 2) Add IAA to each sample tube of cells, sEVs, and sIVs to a final concentration of 40 mM and incubate at room temperature for 1 hour in the dark.

[0278] 3) First, mark the number of the above samples on the collection tube, equilibrate the 10kDa ultrafiltration tube twice with HPLC-grade methanol, each time with a volume of 150μl of methanol, 14,000g for 5min, then add 300μl of 50mM NH4HCO3, then rinse twice, add 100μg of reduced alkylated protein sample, centrifuge at 14000g for 20min at 4℃, add 300μl of 50mM NH4HCO3 and rinse three times, replace the new collection tube, and add 75μl of 50mM NH4HCO3 to the ultrafiltration tube.

[0279] 2.1.3 Protease cleavage

[0280] 1) Add 3 μg of trypsin for mass spectrometry and incubate in a 37°C incubator for 14-16 hours.

[0281] 2) On the next day, centrifuge at 14,000 g for 20 min at 4°C, add 50 μl of 50 mM NH4HCO3, rinse twice, add 1% (volume ratio) formic acid to the collection tube to terminate the enzyme cleavage, and evaporate to dryness in a vacuum at 60°C.

[0282] 2.1.4 Library construction and fractionation

[0283] 1) Resuspend the samples in 30 μl of 0.1% formic acid in water and measure the concentration using a nanodrop. Next, take approximately 10 μg of peptides from each sample and combine them into one sample S.

[0284] 2) 6 μg of sample S was taken out for mass spectrometry analysis in DDA mode, and the remaining S sample was fractionated on a homemade high pH reverse phase column.

[0285] 3) For fractionation, prepare the required reagents according to Table 5. Buffer A is 100% acetonitrile (ACN), and Buffer B is 0.1% trifluoroacetic acid (TFA).

[0286] Table 5 Fractionation reagent ratio

[0287]

[0288] 4) Use a wire to load a layer of C18 membrane into a 200 μl pipette tip. Next, dissolve 30 mg of high pH resistant C18 filler in 200 μl of Buffer A and add the filler to the fractionation column. Then, centrifuge at 3,000 g for 2 minutes at 4°C, add 200 μl of Buffer A to wash once, and then add 200 μl of Buffer B to wash three times. Finally, set the column aside for later use.

[0289] 5) Load the cleaved peptides onto the fractionation column and repeat the loading 5 times.

[0290] 6) Wash the fractionation column three times with 200 μl of Buffer B. Then, add 150 μl of elution buffer of different concentrations and perform gradient elution. Combine the 6% and 35% fractions into one fraction, and take 1.5 μg of peptides from each fraction for mass spectrometry analysis in DDA mode.

[0291] 2.2 HPLC-MS parameters of cells and their sEVs and sIVs

[0292] The peptides after enzyme digestion were loaded onto a homemade Trap column (100 μm × 2 cm, C18 filler, 3 μm particle size, 120A) using phase A (containing 0.1% formic acid, 2% acetonitrile and 97.9% water) at a flow rate of 3 μl / min. Subsequently, the Trap column was eluted using different gradients of phase B (containing 97.9% acetonitrile, 2% water and 0.1% formic acid). These eluted peptides passed through an analytical column (150 μm × 15 cm, C18 filler, 1.9 μm particle size, 120A) to form an electrospray and finally entered the mass spectrometer detector.

[0293] The gradient of phase B was set as follows: 0 min to 5%, 2 min to 10%, 65 min to 22%, 91 min to 35%, 92 min to 80%, 105 min to 80%, 106 min to 5%, 120 min to 5%, and the flow rate was maintained at 500 nL / min throughout the process.

[0294] When performing DDA scanning, the mass spectrometry parameters were set as follows: the accumulation time of TOF MS was 0.25 seconds, the mass scanning range covered 300-1500 Daltons (Da), only ions with valences of +2 to +5 were detected, and the mass deviation was required to be less than 50 ppm. A maximum of 60 ions were monitored in each cycle, and after each detection, the detected ions were isolated for 16 seconds. The fragmentation energy mode used the dynamic fragmentation mode. The accumulation time of the Product ion was 0.04 seconds, and the high-sensitivity scanning mode was used.

[0295] When performing DIA scanning, the mass spectrometry parameters are different: the accumulation time of TOF MS is 0.05 seconds, and the secondary scan uses the high sensitivity mode. The number of variable windows is set to 100, the accumulation time of each window is 30 milliseconds, and the mass scanning range is also 300-1500Da. The specific mass range of each variable window is calculated using the SWATH Variable Window Calculator_V1.1 program.

[0296] 2.3 Cell and sEVs, sIVs protein spectrum data processing and bioinformatics analysis

[0297] The raw data collected in DDA mode were searched using Proteinpilot software (version 5.0.1) with trypsin as the restriction enzyme. The database used was the Uniprot database, which contains 20,431 annotated proteins and was published in July 2019. The screening condition was unused protScore greater than 0.05. The search results of Proteinpilot were imported into SWATH software (version 2.0) as a database to quantify the data collected in DIA mode.

[0298] During the quantitative process, 6 peptides were selected for each protein, and 6 transitions (ion pairs) were selected for each peptide. The confidence of the peptide was set to 99%, and the FDR (false positive rate) was set to 1%. At the same time, modified peptides were excluded, the peak extraction window was set to 10 minutes, and the mass deviation was controlled within 50ppm. Two endogenous peptides were selected every 10 minutes to correct the retention time, and the output peak area was used as the quantitative value.

[0299] The processing of protein expression data involves the following steps: first, the original quantitative values ​​are log2 transformed to meet the normal distribution, and then normalized using the normalize.quantiles function in the preprocessCore package in the R language. After removing proteins without gene names, the stats package in the R language is used for differential analysis, and proteins with a P value less than 0.05 and a change fold greater than 1.5 are screened as differential proteins. At the same time, the corrected p value is set to less than 0.05.

[0300] GO and pathway analysis were performed using the Cytoscape plug-in clueGo. In GO enrichment analysis, cell component (CC), molecular function (MF), and biological process (BP) were selected for analysis. In pathway enrichment analysis, the kegg and reactome databases were selected for analysis.

[0301] Heatmap, principal component analysis (PCA) score, Venn diagram, volcano map, etc. were performed using R language or drawn using Hiplot software. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using Metascape online analysis software.

[0302] 2.4 ELISA

[0303] Collect sEVs and sIVs of MSCs, adjust to equal mass, and operate according to the instructions of the enzyme linked immunosorbent assay (ELISA) kit. Add the sample to the wells coated with antibodies, incubate for 2 hours, and wash the wells; add biotin, incubate for 1 hour, wash the wells; add HRP, incubate for 1 hour, wash the wells; add TMB substrate, incubate for 20 minutes; add STOP solution until the color is obvious. Use an enzyme reader to read the absorbance value of each well in the well plate, using a wavelength of 450nm / 540nm.

[0304] 3. Statistical processing

[0305] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. SPSS22.0 was used to analyze all quantitative data, and t test was used for statistical analysis between the two groups. P Values ​​< 0.05 were considered statistically significant.

[0306] 4. Experimental results

[0307] 4.1 Venn diagram showing differences in protein composition of cells and their sEVs and sIVs

[0308] To characterize the molecular composition of sEVs and sIVs, we performed proteomic analysis of sEVs and sIVs derived from MSCs using label-free mass spectrometry and compared them with cells. In MSCs, 2744 proteins were identified; at the same time, 1678 proteins were detected in sEVs and 2066 proteins were found in sIVs ( Fig.13 sIVs have a greater variety of proteins than sEVs.

[0309] 4.2 Principal component analysis reveals differences in protein composition of cells and their sEVs and sIVs

[0310] PCA was used to analyze the protein components of cells and their sEVs and sIVs ( Fig.14), the results showed that cells, sEVs and sIVs showed different protein distribution patterns. sEVs and sIVs showed significant differences in protein expression. This indicates that sIVs are different from sEVs and have unique protein expression characteristics.

[0311] 4.3 Quantifiable protein differential analysis of cells and their sEVs and sIVs

[0312] Further statistics showed that there were 1425 differentially expressed proteins between sEVs and sIVs in MSC cells, of which 753 were significantly downregulated in sIVs compared with sEVs, 672 were significantly upregulated, 468 of which had a difference of more than 10 times, 227 were significantly downregulated in sIVs compared with sEVs, and 241 were significantly upregulated ( Fig.15 ). This further demonstrates the uniqueness of sIVs.

[0313] The most significantly upregulated and downregulated proteins in sEVs and sIVs were not exactly the same ( Fig.16 ). However, membrane-associated proteins were expressed at lower levels in sIVs, whereas endoplasmic reticulum and ribosome-associated proteins were more abundant in sIVs. This preliminarily suggests that sIVs are unique entities produced by cells, rather than precursors or fragments of cell lysates or extracellular vesicles.

[0314] 4.4 Differences in the expression of exosomal markers in cells and their sEVs and sIVs

[0315] We extracted the list of exosome markers recommended by MISEV2018 (Théry C., Witwer KW, Aikawa E., et al. Minimal information for studies of extracellular vesicles2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines [J]. Journal of extracellular vesicles, 2018, 7(1): 1535750.) and compared sEVs and sIVs with these markers. sEVs showed higher expression levels of exosome markers, while sIVs expressed lower levels of most exosome marker proteins ( Fig.17 ). This further shows that sIVs do not have exosome characteristics and are unique vesicles from inside cells.

[0316] 4.5 Differential expression of organelle marker proteins in cells and their sEVs and sIVs

[0317] We also performed a comparative analysis of the organelle protein expression profiles of sEVs and sIVs. Overall, sIVs contained higher levels of intracellular organelle proteins than sEVs ( Fig.18 In particular, sIVs showed elevated expression levels of proteins associated with membrane-enriched organelles such as endosomes, endoplasmic reticulum, and Golgi apparatus. In contrast, sEVs contained more abundant cellular membrane proteins ( Fig.18 ). This indicates that sIVs have intracellular characteristics.

[0318] 4.6 Differential expression of Clathrin protein family in cells and their sEVs and sIVs

[0319] Among the proteins contained in intracellular vesicles, the Clathrin protein family is essential for the organization and activity of vesicles. Clathrin proteins play a key role in intracellular transport by promoting cargo transport between organelles such as the endoplasmic reticulum, Golgi apparatus, and endosomes in the secretory and endocytic pathways. Given the important role of the clathrin family, we compared the expression levels of clathrin family proteins in sEVs and sIVs. Notably, we observed that most clathrin family proteins were upregulated in sIVs, while sEVs showed low expression ( Fig.19 This finding suggests that the sIVs we isolated may participate in communication between different cellular compartments within the cell.

[0320] 4.7 Enrichment analysis of sIVs-specific proteins

[0321] In Example 4, we analyzed and obtained 106 proteins that are different from sEVs in expression. These proteins are unique proteins expressed by sIVs and can represent the characteristics of sIVs. We performed gene enrichment analysis on these proteins. In terms of cellular components (CC), these proteins are related to COPII-coated endoplasmic reticulum to Golgi transport vesicles, transport vesicles, coated vesicles, endoplasmic reticulum to Golgi transport vesicle membranes, endoplasmic reticulum to Golgi intermediate compartments, transport vesicle membranes, coated vesicle membranes, etc. ( Fig. 20 Meanwhile, in the biological process (BP) category, the terms are glycerophospholipid biosynthesis process, response to ER stress, ubiquitin-dependent ERAD pathway, intracellular protein transport, and ER-to-Golgi vesicle-mediated transport, etc. ( Fig.21 This result indicates that the sIVs we isolated are intrinsic vesicle components in cells.

[0322] 4.8 Comparison of cytokines contained in sEVs and sIVs

[0323] Intracellular vesicles are involved in the intracellular transport of various secretory factors, while exosomes carry these factors outside the cell. Therefore, we compared the levels of cytokines carried by sEVs and sIVs. Proteomic analysis showed that the levels of interleukin-1β (IL-1β) and insulin-like growth factor 2 (IGF-2) were lower in sIVs compared with sEVs ( Fig. 22 ). We then used ELISA to further quantify low-abundance cytokines. The results showed that, based on the same mass, sIVs contained high levels of insulin-like growth factor 1 (IGF-1), epidermal growth factor (EGF), and interleukin-10 (IL-10). Fig.23 ). At the same time, IL-6 ( Fig.23 D) levels were not significantly different, and the levels of tumor necrosis factor α (TNFα) ( Fig.23 E) low levels.

[0324] 5. Summary

[0325] In this example, we used MSCs cells as an example and used proteomics technology to characterize the protein composition of cells, sEVs and sIVs. The results showed that sIVs had a unique protein expression profile, which was different from cells and sEVs; sIVs expressed low exosome markers and highly expressed marker proteins and Clathrin protein family of intracellular membrane-rich organelles, which indicated that sIVs played a role in intracellular material transport and mediated intracellular organelle communication; gene enrichment analysis of sIVs directly suggested that sIVs were involved in the transport of substances between the endoplasmic reticulum and the Golgi apparatus, including forward transport mediated by endoplasmic reticulum to Golgi apparatus vesicles and retrograde vesicle-mediated transport from the Golgi apparatus back to the endoplasm. These findings strongly suggest that sIVs play a key role in the intracellular material transport process, especially mediating the exchange of substances between organelles. Among them, COP-coated vesicles have been widely reported to participate in the intracellular material transport process initiated by the endoplasmic reticulum. During this process, correctly folded and assembled proteins in the endoplasmic reticulum are encapsulated into COP-coated transport vesicles, which then detach from the endoplasmic reticulum membrane. Next, the vesicles shed their coating and fuse with each other to form tubular clusters of vesicles. The Golgi apparatus is responsible for modifying these proteins and lipids received from the endoplasmic reticulum and distributing them to the cell membrane, endosomes, and secretory vesicles. Proteins and lipids move in the cis-to-trans direction within the Golgi apparatus and complete this process through vesicular transport. Proteomic analysis further confirmed that sIVs is involved in the intracellular vesicular transport process and is closely related to the endoplasmic reticulum, Golgi apparatus, and COP-coated vesicles.

[0326] The above results indicate that sIVs are completely different from sEVs, sIVs play an important role in intracellular substance transport, and sIVs are a unique group of vesicles.

[0327] Example 6: sIVs have unique miRNA expression profiles

[0328] 1. Experimental instruments and materials

[0329] 1.1 Experimental Reagents

[0330] Table 6 Experimental reagents

[0331]

[0332] 1.2 Experimental instruments

[0333] Table 7 Experimental instruments

[0334]

[0335] 2. Experimental methods

[0336] 2.1 Data collection and analysis

[0337] 2.1.1 RNA isolation, library preparation, and sequencing

[0338] After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS and tested for RNA degradation and contamination on a 1% agarose gel. RNA purity was checked using a NanoPhotometer® spectrophotometer. RNA concentration was measured using the Qubit™ RNA assay kit in a Qubit® 2.0 Flurometer. The RNANano 6000 assay kit for the Agilent Bioanalyzer 2100 system was used for detection.

[0339] 2.1.2 Library preparation for small RNA sequencing

[0340] 3 μg of total RNA from each sample was used as the input sample for the small RNA library. The sequencing library was generated using the Small RNA Library Prep Set for Illumina® (of NEBNext® Multiplex software, and an index code was added to assign the sequence to each sample. Amplification was performed on a PCR instrument using LongAmp Taq 2X Master Mix, SR Primer for Illumina, and index (X) primers. The PCR product was then purified on an 8% polyacrylamide gel (100 V, 80 min). DNA fragments corresponding to 140-160 bp were recovered and dissolved in 8 μL elution buffer. Finally, the library quality was assessed on an Agilent Bioanalyzer 2100 system using DNA High Sensitivity Chips.

[0341] 2.1.3 Cluster generation and sequencing

[0342] The index-coded samples were clustered using the TruSeq SRCluster Kit v3-cBot-HS (Illumia) on the cBot Cluster Generation System according to the manufacturer’s instructions. After cluster generation, sequencing was performed on the Illumina HiSeq 2500 / 2000 platform to generate 50 bp single-end reads for library preparation.

[0343] 2.1.4 Data Analysis

[0344] 1) Quality Control

[0345] First, the raw data (raw reads) in fastq format were processed by custom Perl and Python scripts. In this step, clean data (clean reads) were obtained from the raw data by removing reads containing ploy-N, 5' end adapter contamination, no 3' end adapter or inserted tags, containing ploy A or T or G or C, and low quality. At the same time, the Q20, Q30 and GC content of the raw data were calculated. Then, a certain length range was selected from the clean reads for all downstream analyses. Bowtie (Langmead B., Trapnell C., Pop M., Salzberg SL Ultrafast and memory-efficient alignment of short DNA sequences to the human genome [J]. Genomebiology, 2009, 10(3): R25.) was used to map the small RNA tags to the reference sequence, without allowing mismatches, to analyze their expression and distribution on the reference sequence.

[0346] 2) Alignment of known miRNAs

[0347] The mapped small RNA tags were used to search for known miRNAs. Using miRBase20.0 as a reference, the modified software mirdeep2 (Friedländer MR, Mackowiak SD, Li N., et al. miRDeep2accurately identifies known and hundreds of novel microRNA genes in sevenanimal clades [J]. Nucleic acids research, 2012, 40(1): 37-52) and srna-tools-cli were used to obtain potential miRNAs and draw secondary structures. Custom scripts were used to obtain the miRNA counts and base biases at the first position of identified miRNAs with a specific length, as well as the miRNA counts and base biases at each position of all identified miRNAs.

[0348] 3) Summary of small RNA annotation

[0349] Summarize all the alignments and annotations obtained previously. In the previous alignments and annotations, some small RNA tags may be mapped to multiple categories. To ensure that each unique small RNA is mapped to only one annotation, we follow the following priority rule: known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>new miRNA.

[0350] 4) Data Analysis

[0351] Target gene prediction was performed using miRanda, and miRNA target gene prediction was the intersection of miRanda and RNAhybrid. The differentially expressed miRNA input data was the readcount data obtained from the miRNA expression level analysis. For samples with biological repeatability: the DESeq R package (3.0.3) was used to perform differential expression analysis on the two conditions / groups. The P value was adjusted using the Benjamini&Hochberg method. By default, the corrected P value was set to 0.05 as the threshold for significant differential expression. Heat maps, principal component analysis, Venn diagrams, volcano maps, etc. were drawn online using Hiplot and adjusted using Adobe Illustrator.

[0352] 5) GO and KEGG enrichment analysis

[0353] GO enrichment analysis was performed on the target gene candidates of differentially expressed miRNAs (hereinafter referred to as "target gene candidates"). GO enrichment analysis used GOseq based on Wallenius non-central hypergeometric distribution, which can adjust gene length bias. KOBAS software was used to test the statistical enrichment of target gene candidates in KEGG pathways.

[0354] 3. Experimental results

[0355] 3.1 Relative RNA abundance in sEVs and sIVs

[0356] First, we analyzed the RNA abundance of sEVs and sIVs using a bioanalyzer ( Fig.24 ).

[0357] 3.2 Distribution of small RNAs in sEVs and sIVs

[0358] Small RNAs associated with cells and extracellular vesicles have been a hot topic in recent years, especially microRNAs (miRNAs), which have multiple biological functions and can be used as biomarkers for multiple diseases. We analyzed the types of small RNAs in sEVs and sIVs. The alignment and annotation of small RNAs of various types with total RNA were summarized. Since there are cases where one sRNA is aligned with several different annotation information at the same time, in order to make each unique sRNA have a unique annotation, small RNAs were classified according to the priority order of known miRNA>rRNA>tRNA>snRNA>snoRNA>YRNA>repeat>gene>novel miRNA detection, and the proportion of each small RNA in total RNA was calculated. The results showed that in sEVs, YRNA is the most important RNA (YRNA is a highly conserved small non-coding RNA (see Xie Yuxin, Chen Tianxing, Wang Li, et al. YRNA: Research Progress in Cancer and Non-Cancer [J]. Chinese Journal of Experimental Surgery, 2021, 38(9): 1844-1848.)); in sIVs, miRNA is the most important RNA. MiRNA accounts for 29.15% in sEVs of MSCs and 92.52% in sIVs. sIVs have more abundant small RNA species, among which the content of miRNA accounts for significantly more than sEVs ( Fig.25 ).

[0359] 3.3 Global miRNA expression characteristics of sEVs and sIVs

[0360] MiRNA has a rich biological regulatory role and accounts for a large proportion of small RNA. Therefore, we conducted a follow-up analysis of miRNA and used a Venn diagram to analyze the types of miRNA contained in sEVs and sIVs. The results showed that 694 miRNAs were detected in sEVs and 989 miRNAs were found in sIVs ( Fig.26 There is some overlap in the types of miRNAs between them, but they are not exactly the same.

[0361] Because sEVs and sIVs contain commonly expressed miRNAs, principal component analysis was subsequently used to compare the miRNA expression patterns of sEVs and sIVs. The results showed that the miRNA components contained in the two vesicles were quite different and the expression patterns were unrelated ( Fig. 27 ), further indicating that sIVs are different from sEVs and contain unique miRNA expression profiles.

[0362] 3.4 Highly Abundant miRNAs in sEVs and sIVs

[0363] By analyzing the top 10 high-abundance miRNAs in sEVs and sIVs, it was found that miR-148a-3p and let-7i-5p were expressed at high levels in sEVs; let-7f-5p was expressed at high levels in sIVs. At the same time, miRNAs such as miR-148-3p, miR-21-5p and miR-100-5p were expressed at high levels in both sEVs and sIVs ( Fig.28 ).

[0364] 3.5 Analysis of differentially expressed miRNAs in sEVs and sIVs

[0365] To further compare the differences in miRNAs between sEVs and sIVs, we performed differential expression analysis on sEVs and sIVs. The results showed that there were 70 differential miRNAs in MSCs between sEVs and sIVs, of which 22 were significantly downregulated in sIVs compared with sEVs, and 48 were significantly upregulated ( Figure 29-30 ). This further illustrates that sIVs are different from sEVs.

[0366] 3.6 Enrichment analysis of differentially expressed miRNA target genes between sEVs and sIVs

[0367] MiRNA exerts its biological effects by regulating downstream target genes. Therefore, after comparing the differential miRNAs in each group, we performed gene enrichment analysis on the target gene sets of these miRNAs, including GO analysis and KEGG analysis. For the sake of convenience, we will refer to "target genes of differentially expressed miRNAs" as "candidate target genes." The results of GO enrichment analysis showed that the candidate target genes of sEVs and sIVs of MSCs were related to intracellular metabolic processes, cell localization was intracellular membrane-related organelles, etc., and molecular functions were related to protein binding and enzyme metabolic reactions, etc. ( Fig.31 A); KEGG pathway analysis showed that candidate target genes of sEVs and sIVs of MSCs were related to pathways such as axon guidance, cell differentiation, endocytosis, and immune regulation (T cell receptor signaling pathway, B cell receptor signaling pathway) ( Fig.31 B).

[0368] 4. Summary

[0369] In this example, we used MSCs cells as the object, and used small RNA sequencing technology to conduct a detailed analysis of the small RNA composition of sEVs and sIVs, and deeply explored the expression pattern of miRNA. After the miRNA in the cell is generated in the nucleus, it is transported to the cytoplasm and participates in the regulation of target genes. Therefore, the miRNA produced in the cell must first perform its biological function by regulating gene expression and participating in various cell biological processes, such as cell proliferation, differentiation and apoptosis. Many miRNAs have been found to be specifically expressed in different types of stem cells, regulating the process of cell differentiation and maturation of specific cell lines. Other miRNAs may promote or inhibit cell death signaling pathways, thereby affecting cell survival and apoptosis. They can maintain cell homeostasis by regulating apoptosis-related genes, such as BCL2 family members, caspase and p53. In cells, miRNA can act as a signaling pathway regulator to adjust cell biological processes, such as growth factor signals, responding to oxidative stress and inflammatory responses. They can target key molecules in specific signaling pathways, thereby affecting the entire signal transduction pathway. Our experimental results show that sIVs are rich in miRNAs, which suggests that sIVs may play a key role in regulating gene expression, cell proliferation, differentiation, growth, apoptosis and signaling pathways, and have great application potential. The results showed that sIVs showed a unique miRNA expression profile, which was significantly different from sEVs, and the miRNA content in sIVs was richer. This finding suggests that sIVs have potential biological regulatory effects and may promote information exchange between different organelles in cells. By performing candidate gene enrichment analysis on differentially expressed miRNAs in sEVs and sIVs, we further found that sIVs are closely associated with intracellular membrane-like organelles. In summary, sEVs and sIVs differ in small RNA components, especially miRNAs. Enrichment analysis of miRNAs further confirmed that sIVs play an important role in intracellular material transport. Compared with sEVs, sIVs contain more diverse miRNAs and may have richer biological functions. These findings provide important clues for a deeper understanding of the function of sIVs in cell biology.

[0370] Example 7: sIVs have unique lipidomic characteristics

[0371] 1. Experimental instruments and materials

[0372] 1.1 Experimental Reagents

[0373] Table 8 Experimental reagents

[0374]

[0375] 1.2 Experimental instruments

[0376] Table 9 Experimental instruments

[0377]

[0378] 2. Experimental methods

[0379] 2.1 Metabolite extraction

[0380] After separation and enrichment of sEVs and sIVs (prepared in Example 2), they were resuspended in PBS. 200 μL of water was added to the container, followed by 480 μL of an extract solution, which was a mixture of MTBE and MeOH in a ratio of 5:1 and contained an internal standard substance. The mixed solution was quickly placed in a liquid nitrogen tank and frozen for 1 minute, then taken out for thawing and mixed by a vortex mixer for 30 seconds to make the solution uniform. The above freezing, thawing and mixing steps were repeated 3 times, followed by ultrasonic treatment in an ice water bath for 10 minutes. The treated sample was allowed to stand at -40°C for 1 hour. Then, the sample was separated by centrifugation at 3000 rpm (centrifugal force 900 × g, radius 8.6 cm) for 15 minutes at 4°C. 300 μL was taken out of the supernatant, transferred to an EP tube, and vacuum dried. 100 μL of the complex solution (DCM: MeOH = 1:1) was added to the dried sample, vortexed for 30 seconds, and ultrasonically treated again in an ice water bath for 10 minutes. Finally, centrifuge at 13,000 rpm (centrifugal force 16,200 × g, radius 8.6 cm) for 15 min at 4 °C, and transfer 75 μL of the supernatant to a sample injection bottle for testing on the instrument.

[0381] 2.2 Metabolite detection

[0382] Vanquish ultra-high performance liquid chromatograph was used, and the target compounds were chromatographically separated using a Waters ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm) liquid chromatography column. For liquid chromatography, phase A was a 40% aqueous solution containing 10 mmol / L ammonium formate and a 60% acetonitrile solution; phase B was a 10% acetonitrile and 90% isopropanol solution containing 50 mL / 1000 mL (10 mmol / L) ammonium formate aqueous solution. We used the following gradient elution program: 0-1.0 min, 40% B; 1.0-12.0 min, linear increase to 100% B; 12.0-13.5 min, maintain 100% B; 13.5-13.7 min, linear decrease to 40% B; 13.7-18.0 min, maintain 40% B. The mobile phase flow rate was set at 0.3 mL / min, the column temperature was 55 °C, the sample tray temperature was 4 °C, and the injection volume was 2 μL (positive and negative ion modes).

[0383] At the same time, the primary and secondary mass spectrometry data were collected using a Thermo Q Exactive HFX mass spectrometer under the control of Xcalibur control software (version: 4.0.27, Thermo). The specific parameters were as follows: Sheath gas flow rate was set to 10Arb, Capillary temperature was set to 350°C, Full ms resolution was set to 120000, MS / MSresolution was set to 7500, Collision energy was set to 10 / 30 / 60 in NCE mode, and Spray Voltage was set to 4 kV (positive ion mode) or -3.8 kV (negative ion mode).

[0384] 2.3 Data Analysis

[0385] ProteoWizard software was used to convert the mass spectra into mzXML format. XCMS was then used for retention time correction, peak identification, peak extraction, peak integration, and peak alignment, with minfrac set to 0.5 and cutoff set to 0.3. Lipid identification was performed using XCMS software, a self-written R package, and the lipidblast database. Bioinformatics graphics were drawn online using Hiplot and adjusted using Adobe Illustrator.

[0386] 3. Experimental results

[0387] The ionization source of the Orbitrap platform is electrospray ionization, which has two ionization modes: positive ion mode (POS) and negative ion mode (NEG). Combining the two modes when detecting metabolomes can achieve higher metabolite coverage and better detection effects. Generally, one ion mode is selected for data analysis. This study takes the positive ion mode as an example for analysis.

[0388] 3.1 Proportion of various metabolites in sEVs and sIVs

[0389] The secondary spectra of lipidomics are accidental, so only lipids identified in all groups in a set of comparative information are credible. Therefore, we classified and counted the metabolites identified in different cells according to the chemical classification information. The proportion of each type of metabolite is as follows: Fig.32 As shown in the figure, 31 species were identified in MSCs. There were differences in the expression levels of various lipids in sEVs and sIVs. PC and PE are common lipid components of cell membranes. Fig.32 The results showed that the proportions of PE and PC in the vesicles were high, indicating the presence of a large number of biomembrane structures in sEVs and sIVs.

[0390] 3.2 Total lipid expression characteristics in sEVs and sIVs

[0391] Metabolomics data has the characteristics of high throughput. The use of principal component analysis can effectively highlight the overall distribution trend of metabolomics data and the degree of difference between samples in different groups. The results showed that MSCs cell sEVs and sIVs have different lipid distribution patterns ( Fig.33 ), that is, sIVs are a unique population of vesicles that are distinct from sEVs and have significantly different lipid expression patterns.

[0392] 3.3 Differential lipid expression characteristics in sEVs and sIVs

[0393] Heat maps can intuitively display the overall distribution of metabolite differences between groups. We visualized the results of screening differential metabolites in the form of heat maps. Fig.34 shown.

[0394] 3.4 Differential lipid content changes and classification information in sEVs and sIVs

[0395] The lipid group bar graph uses the content change degree and classification information of metabolites for visual display. The results of the sIVs group and the sEVs group are as follows: Fig.35 As shown, each column in the lipidome bar graph represents a metabolite. PC, PI, PE, PG, and OxPI were significantly overexpressed in MSCs and in sIVs, among which PC was overexpressed 200 times in sIVs.

[0396] 4. Summary

[0397] Lipidomics identifies and quantifies various lipid molecules. Lipids are divided into eight categories, including fatty acyl, glycerolipids, phospholipids, sterol lipids, propenol lipids, sphingolipids, glycolipids and polyketides. The cell membrane mainly contains various phospholipids, which can be further divided into glycerophospholipids and sphingomyelin, which have obvious differences. Glycerophospholipids are mainly located in the inner leaflet of the phospholipid bilayer in the cell membrane, and together with cholesterol, they constitute the main components of the cell membrane. In this example, sIVs contain more glycerophospholipids, such as PC and PE. Sphingomyelin is a type of phospholipid containing a sphingosine group. Sphingomyelin is located in the outer leaflet of the cell membrane and is mainly involved in neuronal activity and signal transduction. In this example, sEVs contain more sphingomyelin, such as SM. In addition, glycerophospholipids are also involved in many other physiological processes in the body, such as energy metabolism, hormone synthesis, etc.; while sphingomyelin plays a relatively small role in these processes. PC and PE expression levels are high in sIVs, among which PC is more than 200 times higher in sIVs. Among them, PC is also known as lecithin, which is known as the "third nutrient" alongside proteins and vitamins, and plays many important roles in biology. Lecithin can increase the axonal growth of neurons, promote brain development, enhance memory, and prevent Alzheimer's disease. In addition, PE is one of the main molecules that constitute the skeleton of biological membranes. Its unique structure, including a phosphate group, a glycerol, an acyl group, and an ethanolamine, enables it to form stable non-lamellar and multi-layer liposome vesicles in biological membranes. This structure provides a stable foundation for biological membranes and helps maintain the normal structure and function of cells.

[0398] Interestingly, the PC and PE highly expressed by sIVs are both glycerophospholipids. The endoplasmic reticulum is the site of glycerophospholipid synthesis, so it is reasonable that sIVs contain more glycerophospholipids, which further confirms that sIVs are intracellular components that mediate intracellular material transport and communication between organelles. However, sEVs contain more sphingomyelin. sEVs originate from the invagination of the cell membrane and are secreted to the outside of the cell through the cell membrane, so they contain more components outside the cell membrane. This also shows that sIVs lack an external membrane structure, and the difference in multiple lipids distinguishes sIVs from sEVs. Previous studies have shown that the transport of proteins and lipids in cells is related to membrane curvature and lipid distribution effects. Glycerophospholipids can regulate the curvature and fluidity of the membrane by adjusting the chain length and cooperating with cholesterol, giving sIVs more vitality, thereby continuously participating in membrane fusion and fission events in cells.

[0399] In this example, lipidomics data further verified that sIVs are different from sEVs, providing an important basis for in-depth exploration of the unique properties of sIVs in cell and tissue compatibility.

[0400] Example 8: In vitro cultured cells and in vivo retinal tissue have higher absorption efficiency for sIVs

[0401] 1. Experimental instruments and materials

[0402] 1.1 Experimental Reagents

[0403] Table 10 Experimental reagents

[0404]

[0405] 1.2 Experimental instruments

[0406] Table 11 Experimental instruments

[0407]

[0408] 2. Experimental methods

[0409] 2.1 Cell culture

[0410] Human HRMECs were purchased from Angioproteomie, USA. Preparation of complete cell culture medium: Add 5 ml of fetal bovine serum, 1 ml of growth factor and 1 ml of penicillin-streptomycin to 93 ml of ECM basic medium to obtain a complete culture medium containing 5% serum. Preparation of complete culture medium for RPE cells: Add 5 ml of fetal bovine serum and 0.5 ml of penicillin-streptomycin to 44.5 ml of DMEM basic medium to obtain a complete culture medium containing 10% serum. Use a low-power microscope to check the cell growth density, and use a high-power microscope to observe that the cells are round, oval or polygonal flat cells, with rich components in the cytoplasm and small vacuoles in the cells. The cells were inoculated in a culture flask and cultured in a 5% CO2 incubator at 37°C. The culture medium was changed every 3 days. When the cell fusion reached 80%, it was subcultured at a subculture ratio of 1:2, and cells from P3 to P5 were used for experiments.

[0411] 2.2 Experimental animals

[0412] Healthy male C57BL / 6 mice, 8 weeks old, weighing 21-22 g, SPF grade, were purchased from Beijing Weitong Lihua Company (animal production license: SCXK (Beijing) 2016-0006). All experimental animals were fed a normal diet at room temperature, with a light and dark time of 12 h respectively. The animal breeding environment and experimental operations were in accordance with the provisions of the "Regulations on the Administration of Laboratory Animals" of the State Science and Technology Commission, and were approved by the Animal Ethics Committee of this hospital (ethics number: TJYY2019091124). The mice were divided into groups according to the random number table method for subconjunctival and subretinal injections.

[0413] 2.3 Co-culture of DiD-labeled sIVs and sEVs with cells

[0414] Vesicles were incubated with a solution of lipid-soluble tracer DiD for 30 minutes at 37°C. Excess DiD was removed by Amicon® Ultra centrifugal filters. Cells were seeded into pre-placed circular pieces in 24-well culture plates and incubated for 24 hours. DiD-labeled vesicles were added to the culture medium of the cells and incubated at 37°C for different times: 3 hours, 12 hours, 24 hours, or 48 hours. After removing the culture medium, the cells were washed with PBS at room temperature. Cells were fixed with 4% PFA for 10 minutes and then incubated with 0.1% Triton X-100 for 5 minutes at room temperature to permeabilize the cells. Subsequently, cells were stained with CoraLite® Plus488-conjugated Phalloidin antibody for 30 minutes at room temperature. Cell nuclei were labeled with DAPI. Finally, cells were imaged using confocal laser scanning microscopy and the amount of endocytosed vesicles was analyzed by Image J.

[0415] 2.4 Subconjunctival and intravitreal injection of DiD-labeled sIVs and sEVs

[0416] Vesicles were stained with DiD as described above. DiD-labeled vesicles were injected into the subconjunctiva of mice at a volume of 5 μL and a mass of 3 μg. Eyeballs were collected for observation 24 hours and 48 hours after subconjunctival injection. DiD-labeled vesicles were injected into the vitreous cavity of mice at a volume of 1 μL and a mass of 1 μg. Eyeballs were collected for evaluation 8 hours and 48 hours after vitreous injection.

[0417] 2.5 Retinal sections

[0418] Frozen retinal sections (8 μm thick) were used for immunofluorescence analysis. Retinal sections were fixed with PFA for 15 minutes at room temperature, then the slides were washed with PBS and the cell nuclei were stained with DAPI. Finally, the slides were mounted with anti-fluorescence attenuation mounting medium and observed using a confocal laser scanning microscope.

[0419] 3. Statistical processing

[0420] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. SPSS22.0 was used to analyze all quantitative data. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. Nonparametric tests were used for non-normally distributed data and data with unequal variance. P Values ​​< 0.05 were considered statistically significant.

[0421] 4. Experimental results

[0422] 4.1 Cells have a better ability to internalize sIVs than sEVs

[0423] In order to evaluate the phagocytic ability of cells for the two types of vesicles, RPE cells and HRMECs were selected and co-cultured with these MSC-sEVs (referred to as sEVs in this example) and MSC-sIVs (referred to as sIVs in this example). Briefly, equal amounts of DiD-labeled vesicles were incubated with cells, and the distribution of DiD was observed using confocal microscopy at different time points to observe the amount of vesicle uptake by cells. The results showed that after 3 hours of co-culture, cells began to internalize sEVs and sIVs, and their uptake continued to increase over time ( Fig.36 A and B, Fig.37 A and B). The amount of sEVs and sIVs internalized by cells reached a peak at 24 hours and then gradually decreased within 24-48 hours ( Fig.36 C, Fig.37 C). Notably, in both RPE cells and HRMECs, the internalization rate of sIVs consistently exceeded that of sEVs from 12 to 48 h ( Fig.36 C, Fig.37 C).

[0424] 4.2 The ability of retinal internalization of sIVs is superior to sEVs

[0425] To evaluate the phagocytic capacity of the retina for both types of vesicles, an equal amount of DiD-labeled vesicles was administered to the eye by two methods: subconjunctival injection and intravitreal injection. Eyeball samples were collected at different time points, and then frozen sections were prepared and confocal microscopy was used to observe vesicle uptake within the retina. The results showed that 24 hours after subconjunctival injection, sIVs penetrated the sclera and entered the subretinal and retinal spaces, while sEVs accumulated between the conjunctiva and sclera, with fewer sEVs reaching the subretinal area ( Fig.38 A). After 48 hours, the retina showed significant uptake of sEVs and sIVs ( Fig.38 C). Specifically, sEVs were distributed in the RPE layer, while sIVs were widely distributed in the outer nuclear layer, inner nuclear layer, and ganglion cell layer. After intravitreal injection of DiD-labeled vesicles, the vesicles spread from the vitreous cavity to the retina. The results showed that 8 hours after injection, sEVs were distributed in the vitreous cavity and had not yet reached the retina, while sIVs had reached the entire retinal layer and were internalized by cells in the ganglion cell layer and inner nuclear layer, and higher sIVs accumulation was observed on the RPE layer ( Fig.38 B). 24 hours after intravitreal injection, the retina efficiently absorbed sEVs and sIVs, and both types of vesicles were diffusely distributed throughout the retina ( Fig.38 B). sIVs showed significantly more uptake compared to sEVs ( Fig.38D) and is widely distributed in all retinal layers, including the ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and RPE layer ( Fig.38 B).

[0426] 5. Summary

[0427] This example shows that cells cultured in vitro have better endocytosis of sIVs than sEVs. Subconjunctival injection and intravitreal injection in mice showed that the retina had better endocytosis of sIVs than sEVs, which fully demonstrated that sIVs have good tissue compatibility.

[0428] Example 9: Protective effect of sIVs on retinal RGCs and retinal structure in ONC mice

[0429] 1. Experimental methods

[0430] 1.1 Establishment of the mouse optic nerve crush (ONC) model

[0431] After the mice in the ONC group were anesthetized, oxybuprocaine hydrochloride eye drops were used for surface anesthesia of the eyeball. Under the field of view of the surgical dissecting microscope, a pair of spring scissors was used to cut the conjunctiva of one eye at about 4 o'clock on the temporal side of the eyeball, and the orbital muscles were gently deflected to expose the white optic nerve. The optic nerve was clamped for about 5 seconds at a distance of 2 mm from the eyeball. After the crush injury was completed, the incision was sutured. The inclusion criteria of the postoperative model were as follows: the mouse lens had no turbidity; the retina had no hemorrhage or detachment; the fundus blood supply was normal; the eyeball muscles recovered normally, the retraction was normal, there was no protrusion, and it could rotate freely.

[0432] The groups included normal control group (normal group, only optic nerve was exposed without clamping), ONC group + PBS injection into the vitreous after injury, ONC group + sEVs injection into the vitreous after injury, and ONC group + sIVs injection into the vitreous after injury.

[0433] 1.2 Immunofluorescence staining of retinal flat mounts

[0434] The mouse eyeball was removed and fixed in paraformaldehyde at room temperature for 15 minutes, placed in cold PBS for 5-10 minutes, and the eyeball was transferred to gauze. An incision was made at the corneoscleral edge under an operating microscope, and the sclera was gently torn open with two toothed forceps to remove the lens. The retina was cut into four petals with small scissors, and the obvious blood vessel clusters were cut off. Cold anhydrous methanol was gently dripped on the retina for fixation. After the retina turned white, it was transferred to a 2 ml flat-bottom EP tube.

[0435] The anhydrous methanol in the EP tube was aspirated, and the slides were washed twice with PBS on a 4°C shaker. 1 ml of blocking solution was added and the slides were shaken at 4°C for 2 hours. The primary antibody was added and incubated in a 4°C refrigerator for 24 hours (the dilution concentration of the primary antibody RBPMS was 1:200); the slides were washed 3 times, 10 minutes each time; the fluorescent secondary antibody was incubated for 4 hours (the dilution concentration of the secondary antibody Dylight488 was 1:1000); after washing, the slides were sealed with an anti-fluorescence quencher. The slides were observed and photographed under a fluorescence microscope. Three photos of each retina far from the optic disc were selected. The StarDist2D plug-in in ImageJ (v153) software was used to count the number of RGCs in each photo and calculate the average density of RGCs.

[0436] 1.3 Optical Coherence Tomography (OCT)

[0437] After anesthesia and dilation of the pupil, transparent eye gel was applied to the cornea of ​​both eyes for imaging and analysis, and the cornea was kept moist during the whole process. The British Optoprobe was used to capture and measure the mouse retinal images around the optic nerve head. The thickness of the RGCs complex (retinal ganglion cell complex, GCC) obtained by measurement was used as the measurement result, including RNFL, ganglion cell layer (GCL) and inner plexiform layer (IPL). The built-in software was used to segment the GCC and quantify its thickness.

[0438] 1.4 Hematoxylin-Eosin Staining (HE)

[0439] The mouse eyeballs were removed, fixed and paraffin sections were prepared for routine HE staining. The extent of retinal tissue damage in mice was recorded under an optical microscope, and the number of RGC cells was further statistically analyzed.

[0440] 1.5 TUNEL staining of retinal cryosections

[0441] The eyeballs of mice in the experimental group and the control group were taken, and 10 discontinuous frozen sections were taken from the peripheral and central parts, respectively, with the optic nerve as the central reference point, and TUNEL staining was performed according to the instructions of the apoptosis kit. 3-4 fields of view of each section were photographed using a fluorescence microscope, and the number of TUNEL-positive cells in the sections was calculated using computer software to evaluate the apoptosis of cells in the retina of mice in different groups.

[0442] 1.6 Western blotting

[0443] The eyeballs of the test mice in each group were collected and the retinas were separated. The total protein in the retina was extracted, and the protein expression levels of inflammation-related factors (GFAP and iba1), anti-apoptotic protein Bcl-2, pro-apoptotic protein Bax, caspase-3 and c-caspase-3 were detected by Western Blot.

[0444] 2. Statistical processing

[0445] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, and P values ​​< 0.05 were considered statistically significant.

[0446] 3. Experimental results

[0447] 3.1 Successful construction of ONC mouse model

[0448] The ONC mouse optic nerve was clamped for different lengths at 2 mm from the posterior pole, and retinal flat-slides were taken 21 days after modeling to count the number of retinal ganglion cells. The results showed that a large number of retinal ganglion cells were lost after clamping for different lengths, and the degree of damage was similar after clamping for 3 seconds, 5 seconds, and 8 seconds. The ONC mouse model was successfully established, and a clamping time of 3 seconds was selected for later experiments ( Fig.39 ).

[0449] 3.2 sIVs significantly reduced the loss of retinal ganglion cells in ONC mice

[0450] ONC mice were injected with different concentrations of sIVs and sEVs through the vitreous cavity. After 21 days of administration, the number of retinal ganglion cells was stained and counted by retinal flat mount. The results showed that the therapeutic effects of sIVs and sEVs at low concentrations (1.25 mg / ml) were not significantly different from those of the PBS group injected into the vitreous cavity. At high concentrations (2.5 mg / ml), sIVs reduced the loss of retinal ganglion cells. Therefore, high concentrations of sIVs and sEVs were selected for subsequent experiments ( Fig.40 ).

[0451] The retina of ONC mice in each treatment group was flat-mounted, and the number of retinal ganglion cells was counted at different time points after treatment. The results showed that sIVs reduced the loss of retinal ganglion cells 7 days after intravitreal injection, with the same short-term therapeutic effect as sEVs; and sIVs had a better therapeutic effect 21 days after administration, and could maintain the number of retinal ganglion cells for a longer period of time ( Fig.41).

[0452] 3.3 sIVs maintain the thickness of the retinal ganglion cell complex in ONC mice

[0453] The ONC mice were subjected to full retinal optical coherence tomography 21 days after administration to analyze the changes in the structure and thickness of each retinal layer. The results showed that intravitreal injection of sIVs and sEVs had no effect on the morphology and light reflectivity of the retinal layers. The ganglion cell complex (GCC) includes the nerve fiber layer, ganglion cell layer, and inner plexiform layer, which reflect the state of nerve damage. sIVs better slowed down the reduction of GCC thickness and had a better therapeutic effect ( Fig.42 ).

[0454] 4. Summary

[0455] In retinal flat-mount staining 7 days after ONC mouse model treatment, sEVs and sIVs inhibited ganglion cell apoptosis to the same extent. Notably, in whole-retinal optical coherence tomography 21 days after ONC mouse model treatment, sIVs better slowed the reduction of GCC thickness and had a better therapeutic effect.

[0456] Example 10: Neuroprotective effect of MSC-sIVs on stroke mice

[0457] 1. Experimental reagents

[0458] Table 12 Experimental reagents

[0459]

[0460] 2. Experimental methods

[0461] 2.1 Establishment of mouse stroke model

[0462] The mouse photochemical embolism model was induced by rose bengal. Rose bengal is a light-sensitive dye that can react with vascular endothelial cells under light to produce lipid peroxidation, release free radicals, damage endothelial cells, induce platelet adhesion, and form thrombi. In order to better simulate the acute ischemic stroke model, we intervened 7-8 week old C57 male mice with a specific cold light source. The specific steps are as follows:

[0463] (1) Anesthesia: Use 3.5% isoflurane to induce inhalation anesthesia in mice. After successful anesthesia, fix the mice in a prone position on the experimental operating table. After handling the hair on the head of the mice, perform local head skin disinfection. After disinfection, use ophthalmic surgical scissors to cut the skin along the midline of the mouse's head gently to fully expose the stroke window (based on the mouse's anterior fontanelle, 2 mm beside the sagittal suture).

[0464] (2) Light intervention: After completing the above preparations, 75 mg / kg of rose bengal was injected intraperitoneally into the mice. Because rose bengal is toxic, the injection should be slow and completed within 5 minutes. Then a 4 mm diameter optical fiber was fixed at the stroke window, and a green cold light source was used to illuminate this area for 10 minutes at the maximum intensity. During this period, the changes in the vital signs of the mice were noted.

[0465] (3) Suturing: After 15 minutes of irradiation, the scalp wound of the mouse was sutured with medical sutures, and the mouse was returned to the cage after suturing. During the entire surgical process and after surgery, the body temperature of the mouse was always maintained at around 37°C using a mouse electric blanket. In order to reduce the error caused by experimental modeling, all the above experimental steps were completed by the same experimenter on the same day.

[0466] (4) Animal grouping: After the model mice woke up, they were carefully observed for half an hour, and stroke mice with similar vital signs were randomly divided into PBS group, sEVs group, and sIVs group.

[0467] 2.2 Behavioral testing

[0468] Two behavioral test methods, the fatigue transfer rod test and the forelimb grip test, were used to evaluate the degree of recovery of motor function. The fatigue transfer rod test is mainly used to evaluate the motor function and balance and coordination ability of animals; the forelimb grip test is mainly used to evaluate the neuromuscular ability of the animal's forelimbs.

[0469] 2.2.1 Rotarod test

[0470] During the behavioral test, in order to select mice that can adapt to the fatigue transfer rod exercise under the same conditions, all mice were pre-trained on the fatigue transfer rod for 2 days before surgery. The rotarod test is to place the experimental animals on a rotating rod. They need to adjust their center of gravity to maintain balance and avoid falling off the rod. The length of time the experimental animals stay on the rotating rod is measured by adjusting the speed, angle and time of the rotating rod to evaluate the movement and balance ability of the experimental animals. MSC-sEVs (PBS solvent) with a concentration of 1.5μg / μL and a total volume of 20μL, MSC-sIVs (PBS solvent) and an equal volume of PBS were given by nasogastric administration on the day after stroke surgery and on the first, second and third days after surgery, and the rotarod test was performed on the first, third, fifth and seventh days after surgery.

[0471] 2.2.2 Grip test

[0472] The forelimb grip test is used to test and evaluate the neuromuscular function of the forelimbs. The tension sensor tests the forelimb grip. When the mouse forelimbs grasp the grip bar on the grid, the tail is gently pulled back. The grip reaches the maximum value at the moment the mouse forelimbs are released. The bio-function signal system software records the data. Each mouse is tested three times, and the average value is taken for statistical analysis.

[0473] After the behavioral test, the mice were subjected to anatomical and pathological examinations. The specific experimental procedures were as follows:

[0474] 2.3 TTC staining of mouse brain tissue

[0475] TTC staining utilizes the principle of redox reaction. TTC can undergo a specific chemical reaction with succinate dehydrogenase in cells. After cerebral infarction occurs, the succinate dehydrogenase in cells in the infarcted area decreases significantly, and thus cannot fully react chemically with TTC. After the redox reaction occurs, the brain tissue in the infarcted area can be stained white, while the brain tissue in the non-infarcted area can appear red after staining due to the full redox reaction. Therefore, the color change of brain tissue can clearly distinguish between the infarcted area and the non-infarcted area. We usually calculate the volume of brain tissue in the white area, which is the volume of cerebral infarction. The specific steps are as follows:

[0476] (1) Brain removal: After the neurological function evaluation experiment of stroke mice is completed, the stroke mice are deeply anesthetized with chloral hydrate. The PBS solution is placed in the refrigerator in advance. After the mice are fully anesthetized, ice PBS is used for cardiac perfusion until the lungs and liver of the mice are white (in order to speed up the perfusion, the lungs and liver can be cut in advance). After cardiac perfusion, the skull of the mouse is quickly removed, the intact brain tissue is removed, and the removed brain tissue is quickly placed in a -20℃ refrigerator (about 10 minutes), and then the mouse brain is cut into 2mm thick coronal brain slices on ice.

[0477] (2) TTC staining: Prepare 1.5% TTC solution in advance. Soak the above-cut coronal brain slices in TTC solution for sufficient chemical reaction. The reaction conditions are to place them in a 37°C constant temperature incubator away from light for 20 minutes. During the placement period, turn the brain slices over several times to allow for sufficient redox reaction, so that better staining can be achieved. After staining, fix the brain slices in 4% paraformaldehyde for 30 minutes, then place the fixed brain slices on a black background with a ruler next to them, and then take pictures.

[0478] 2.4 TUNEL assay for neuronal apoptosis

[0479] In order to evaluate the effects of sIVs and sEVs treatment on neuronal apoptosis in stroke mice, we used the TUNEL method to detect neuronal apoptosis in this study. The main principle of the TUNEL method for detecting cell apoptosis is that during cell apoptosis, the double-strand breaks of chromosome DNA will produce a large number of sticky 3'-OH ends, and under the action of terminal deoxyribonucleotide transferase (TdT), deoxyribonucleotides and fluorescent substances can be labeled to the 3'-OH end of DNA, thereby enabling the detection of apoptotic cells. The specific steps are as follows:

[0480] (1) Brain extraction: Stroke mice were deeply anesthetized with chloral hydrate and perfused by heart with iced PBS. The perfusion was successful when the lungs and liver turned white. After the perfusion, the skull of the mouse was quickly removed and the brain tissue of the mouse was completely removed. The brain tissue was then immersed in 4% paraformaldehyde solution and kept in a refrigerator at 4°C overnight.

[0481] (2) Dehydration: After the brain tissue is fixed overnight with 4% paraformaldehyde, the residual formaldehyde liquid on the brain tissue is blotted dry. Prepare 15% sucrose aqueous solution and 30% sucrose aqueous solution in advance. Soak the brain tissue in a centrifuge tube containing 15% sucrose aqueous solution for initial dehydration. When the brain tissue completely sinks to the bottom of the centrifuge tube, place the brain tissue in a centrifuge tube containing 30% sucrose aqueous solution for further dehydration. When the brain tissue completely sinks to the bottom of the centrifuge tube again, remove the brain tissue and blot the residual sucrose solution on the brain tissue for the next step.

[0482] (3) Embedding: Prepare the items needed for embedding (tweezers, OCT compound, embedding box, blade, -80℃ ultra-low temperature refrigerator) in advance. Use a pre-refrigerated blade to cut off brain tissues that are not needed later, such as the brain stem and cerebellum. Slowly squeeze the OCT compound into the embedding box (to avoid bubbles that affect subsequent sectioning) so that it slowly covers the bottom of the embedding box. After slowly placing the brain tissue in the middle of the embedding box, continue to slowly add the OCT compound until the OCT compound covers the entire brain tissue. Finally, place the embedding box containing the OCT compound and brain tissue into a -80℃ ultra-low temperature refrigerator to promote OCT fixation through low temperature. At the same time, the embedded brain tissue can be stored in a -80℃ ultra-low temperature refrigerator.

[0483] (4) Slicing: When the temperature in the freezing microtome operating room drops to -20°C, fix the embedded brain tissue. First, perform preliminary trimming of the brain tissue. After the brain layer containing the cerebral infarction lesion is seen, adjust the slice thickness to 8um for slicing. Use a poly-lysine-coated slide to stick the stretched brain slices, and then place the cut brain slices in a slice box. Finally, the slice box can be placed in a -20°C refrigerator for storage.

[0484] (5) Antigen retrieval: Take the brain slices to be stained out of the -20°C refrigerator in advance and place them at room temperature for 30 minutes for antigen retrieval.

[0485] (6) Fixation: After the appropriate area of ​​the frozen section is circled with an immunohistochemistry pen, 4% paraformaldehyde is added for fixation at room temperature for 30 minutes, and then washed with PBS three times, each time for 5 minutes.

[0486] (7) Promote penetration: Add 0.3% TritonX-100 to the slices for 10-20 min at room temperature to promote penetration, and wash three times, 5 minutes each time.

[0487] (8) TUNEL reagent incubation: After the permeation is completed, wash the brain slices with PBS solution for 5 minutes again, and repeat this washing 5 times. Prepare the TUNEL reaction mixture in advance. Cover the brain slices with the TUNEL reaction mixture and place them in a 37°C constant temperature incubator away from light for 1 hour. After the incubation, soak the brain slices in PBS and quickly wash them on a shaker for 5 minutes. Repeat this 5 times. After washing, cover the brain slices with DAPI and seal them with a coverslip. After sealing, observe them immediately under a fluorescence microscope. After obtaining the image data, use Image software to analyze the acquired images.

[0488] 2.5 Immunofluorescence staining to observe microglial activation in each group of stroke mice

[0489] In order to observe the changes in the number and morphology of microglia in different groups after stroke, immunofluorescence staining was used to mark the microglia in the injured area of ​​each group using IBA1, and it was found that microglia were significantly activated after stroke.

[0490] The preparation, fixation and permeabilization steps of brain tissue immunofluorescence frozen sections were the same as those described in Example 1.3.

[0491] (1) Blocking: After fixation and permeation promotion, blocking solution (PBST + 5% normal goat serum + 2% BSA) was added to the frozen sections of brain tissue and placed in a humidified box in the dark for 1.5 h.

[0492] (2) Antibody incubation: Add 1:100 Iba-1 antibody and incubate overnight in a 4°C refrigerator.

[0493] (3) The next day, remove the plate and wash it with PBST three times, 5 min each time. Add 1:300 Alexa Fluor488-labeled goat anti-rabbit secondary antibody and incubate it in a wet box at room temperature for 2 h in the dark. Wash it with PBS three times, 5 min each time.

[0494] (4) Counterstaining the cell nucleus: Add DAPI staining solution and react at room temperature in the dark for 10 min. Wash off the DAPI staining solution with PBS, seal the slides with anti-fluorescence quencher, and store the slides in the dark.

[0495] (5) Observation: Microglial activation was observed using an Olympus fluorescence microscope. A 10× field of view was set for each slice, and at least three areas were randomly selected for photography.

[0496] 2.6 Immunofluorescence staining to observe astrocyte activation in each group of stroke rats

[0497] Related literature (see, Ni XC, Wang HF, Cai YY, et al. Ginsenoside Rb1inhibits astrocyte activation and promotes transfer of astrocyticmitochondria to neurons against ischemic stroke[J]. Redox biology, 2022, 54:102363.;Hasel P, Rose IVL, Sadick JS, et al. Neuroinflammatory astrocyte subtypes in the mouse brain[J]. Nature neuroscience, 2021, 24(10): 1475-1487.) reported that astrocytes are reactively activated in an acute ischemic stroke model, and the effect of related treatments on reducing cerebral infarction volume is closely related to inhibiting the activation of astrocytes. The preparation of brain tissue immunofluorescence frozen sections and immunofluorescence staining steps are the same as those described in Examples 1.3 and 1.4.

[0498] 2.7 Immunofluorescence staining to observe the expression of β-tubulin III in the brain tissue of stroke rats in each group

[0499] The brain tissue was fixed in 4% paraformaldehyde, and then frozen and incubated with antibodies, the steps were the same as those described in Examples 1.3 and 1.4, and images were collected and analyzed using ImageJ software to analyze the immunofluorescence images and calculate the fluorescence intensity of the immunofluorescence images. β-tubulin III is a recognized neuron-specific marker and is commonly used to identify the proliferation and differentiation of neural stem cells.

[0500] 2.8 Immunofluorescence staining to observe the expression of occludin and ZO-1 in the brain tissue of stroke mice in each group

[0501] Brain microvascular endothelial cells actively interact with extracellular matrix proteins that form the basement membrane, pericytes, astrocytes, microglia, and neurons to form the neurovascular unit, which couples neuronal activity to vascular function by controlling regional cerebral blood flow and blood-brain barrier parameters. Ischemic stroke triggers rapid neurovascular unit damage and impairs neurovascular coupling. Oxygen and nutrient deprivation activate proteolytic enzymes, and brain microvascular endothelial cells secrete matrix metalloproteinases (MMPs), which lead to the degradation of tight junction proteins of brain microvascular endothelial cells and increase the permeability of the blood-brain barrier. We detected the expression of CD 31, a key marker of microvascular endothelial cells, also known as platelet endothelial cell adhesion molecule-1 (PECAM-1), in the infarcted area of ​​stroke by immunohistochemical staining.

[0502] 3. Statistical processing

[0503] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, and P values ​​< 0.05 were considered statistically significant.

[0504] 4. Experimental results

[0505] MSC-sIVs improve motor coordination and neuromuscular ability in stroke mice

[0506] The rotarod fatigue test was used to evaluate the motor coordination of mice. Fig.43 As shown in the figure, the time of falling from the rotating rod test in stroke mice was significantly earlier than that in the MSC-sEVs group and the MSC-sIVs group. The time of falling from the rotating rod in the MSC-sEVs group and the MSC-sIVs group was higher than that in the PBS group at different time points. Therefore, the results of the rotating rod test indicate that MSC-sIVs can improve the movement and coordination ability of experimental animals after ischemic stroke and play a neuroprotective role.

[0507] The forelimb grip test is used to test and evaluate the neuromuscular function of the forelimb. Fig.44 As shown, ischemia induced a decrease in forelimb grip strength at various time points within 7 days after ischemia, while administration of MSC-sIVs and MSC-sEVs significantly increased forelimb grip strength 3 days after ischemia.

[0508] The study conducted fatigue transfer rod test and forelimb grip test within 7 days after modeling. The results showed that the motor ability of mice decreased significantly on the day of modeling, which may be related to impaired cerebral cortical motor function and surgical anesthesia. After that, the motor ability of mice gradually recovered, and the MSC-sIVs intervention group recovered more significantly than the PBS group.

[0509] 4.2 Effects of MSC-sIVs on brain tissue of stroke mice

[0510] The TTC staining results can visually show the cerebral ischemic infarction area of ​​each group of mice. The normal brain tissue is red, the white area is the infarction focus, and the red and white brain tissue area is the penumbra area. After TTC staining, it can be seen that the area of ​​the white infarction area in the PBS group is the largest, which is significantly different from MSC-sEVs and MSC-sIVs ( Fig.45 ).

[0511] 4.3 Effects of MSC-sIVs on neural cells in stroke mice

[0512] TUNEL staining data showed that compared with the PBS group, the MSC-sIVs and MSC-sEVs treatment groups significantly reduced the number of neuronal apoptosis in the cerebral cortex of photochemical stroke mice, and the difference was statistically significant compared with the PBS group (P < 0.05) ( Fig.46 ). The above research results show that MSC-sIVs can significantly reduce neuronal pathological damage and the relative number of apoptotic cells in mice after stroke.

[0513] Effects of MSC-sIVs on microglia in stroke mice

[0514] In the ischemic area of ​​stroke mice, the number of activated microglia increased significantly. The number of activated microglia in the cortical ischemic area in the MSC-sIVs and MSC-sEVs groups was reduced compared with the PBS group (P < 0.05). Compared with MSC-sEVs, the intervention of MSC-sIVs significantly reduced the number of activated microglia ( Fig.47 ). The above results indicate that MSC-sIVs can reduce the activation of microglia in the ischemic area after stroke in mice and alleviate the inflammatory stress on neural tissue.

[0515] Effects of MSC-sIVs on astrocytes in stroke mice

[0516] GFAP is a marker of astrocytes, reflecting the degree of damage to the central nervous system. We detected the expression of GFAP, a marker of astrocyte activation, to study the effect of MSC-sIVs on astrocyte activation. Immunofluorescence staining experiments showed that the expression of GFAP protein in the brain tissue of mice in the PBS group was significantly higher than that in the MSC-sIVs group and the MSC-sEVs group. MSC-sIVs can reduce GFAP expression and the effect is more significant than that of MSC-sEVs ( Fig.48 ). Administration of MSC-sIVs after mouse stroke modeling can inhibit the activation of astrocytes in the brain tissue of photochemically induced ischemic stroke model mice.

[0517] Effect of MSC-sIVs on β-tubulin III protein expression in brain tissue of stroke mice

[0518] Immunofluorescence staining experiments showed that the expression of β-tubulin Ⅲ protein in the mouse brain tissue was significantly reduced after modeling, and MSC-sIVs could increase the expression of β-tubulin Ⅲ and the effect was more significant than that of MSC-sEVs ( Fig.49 ).

[0519] 4.7 Effects of MSC-sIVs on the expression of occludin and ZO-1 in the brain tissue of stroke mice

[0520] We used double immunofluorescence staining of tight junction proteins Occludin and ZO-1 to measure the permeability of the blood-brain barrier. Fig.50 As shown in the figure, the expression of Occludin and ZO-1 in the brain tissue of mice in the PBS group was significantly reduced. Compared with the PBS group, the levels of Occludin and ZO-1 in the brain tissue were significantly increased after MSC-sIVs and MSC-sEVs treatment. This indicates that MSC-sIVs may contribute to angiogenesis after ischemic injury in stroke. Immunofluorescence staining showed that the fluorescence intensity of CD31 in the cortex was significantly reduced after stroke, and the co-localization of CD31, Occludin and ZO-1 was also reduced. After treatment with MSC-sEVs and MSC-sIVs, the fluorescence signal was enhanced. However, the group treated with MSC-sIVs had stronger fluorescence signals of Occludin and ZO-1 in the cerebral cortex, and there were more co-localized fluorescent spots.

[0521] 5. Summary

[0522] TUNEL immunofluorescence staining results of ischemic stroke mice showed that both MSC-sEVs and MSC-sIVs could inhibit the apoptosis of ganglion cells. It is worth noting that compared with MSC-sEVs, MSC-sIVs could slow down the activation of astrocytes and microglia and increase the expression of β-tubulin Ⅲ to a greater extent, and had a better therapeutic effect.

[0523] Example 11: Neuroprotective effect of MSC-sIVs on Alzheimer's disease mice

[0524] 1. Experimental reagents

[0525] Table 13 Experimental reagents

[0526]

[0527] 2. Experimental methods

[0528] 2.1 Experimental animals and groups

[0529] Alzheimer's disease (AD) model mice were selected from 4-month-old 5xFAD transgenic mice with C57 / BL6J as the strain background, purchased from Nanjing Junke Biotechnology Co., Ltd. The experiment began after 2 weeks of adaptive feeding. The mice were treated by nasogastric administration every other day for a total of two months; MSC-sEVs (PBS solvent) with a concentration of 1.5μg / μL and a total volume of 20μL, MSC-sIVs (PBS solvent) and an equal volume of PBS were given by nasogastric administration, and the Normal group was not given any drug treatment except hyaluronidase. Hyaluronidase (100U / 5μL) was given intranasally to each mouse before administration to promote better absorption of the drug.

[0530] 2.2 Water maze experiment

[0531] The Morris water maze is a cognitive behavioral experiment designed to assess the learning and memory abilities of rodents in terms of spatial location. The experimental device is a circular pool with a radius of 60 cm and a height of 50 cm. The device is evenly divided into four quadrants. Cards of different shapes and colors are affixed to the pool walls of each quadrant for easy positioning and identification. A camera is installed above the pool, and its field of view can cover the entire pool. Through the water maze image video acquisition system, every detail of the experiment can be accurately recorded. In order to eliminate the influence of external factors such as light on the experimental results, the area around the pool is closed with blackout curtains. During the experiment, about 30 cm of water will be injected into the pool, and the water temperature will be precisely controlled at 21.2℃. In the third quadrant of the pool, a cylindrical platform with a diameter of 10 cm will be placed, which will be submerged 1 cm below the water surface.

[0532] The Morris water maze experiment consists of two parts: the navigation test is used to evaluate the learning ability of mice, that is, how they find the platform hidden under the water through trial and error; the spatial exploration test is used to evaluate the spatial memory ability of mice, that is, whether they can find the original location of the platform by relying on their previous memory after the platform is removed. The specific test steps are as follows:

[0533] 1) Navigation test: A different quadrant was selected every day, and we slowly put the experimental animals into the water facing the pool wall. The time required for the mouse to find the platform hidden under the water surface in the water will be recorded in detail, and this time is defined as the escape latency. Once the mouse successfully finds the platform, it will get a 10-second rest period. However, if the mouse cannot find the platform within 60 seconds, the escape latency will be recorded as 60 seconds. At this time, we will gently guide the mouse to the platform and allow it to stay on the platform for 10 seconds in the hope of enhancing its memory of the platform. After completing the above steps, the mouse will be gently dried with a towel and briefly baked with an incandescent lamp before returning it to the cage. This training process will last for 5 days, with 4 training sessions fixed at a specific time period every day, and the interval between each training session is about 20 minutes. In order to more accurately evaluate the spatial learning ability of mice, the average of the four escape latencies is calculated and statistically analyzed as the performance of the day. This method enables us to have a more comprehensive understanding of the ability of mice in spatial learning.

[0534] 2) Spatial exploration test: 24 hours after completing the navigation test, remove the hidden platform in the third quadrant of the pool. Then, put the mouse into the water from the first quadrant of the pool, and pay close attention to and record the swimming route and direction of the mouse in the pool within 60 seconds after entering the water, as well as the frequency of the mouse crossing the original platform position. In addition, it is necessary to calculate the proportion of time the mouse swims in different quadrants. Through these observations and analyses, we can understand the spatial memory ability of mice in many aspects.

[0535] 2.3 Open field experiment

[0536] The open field test is a method used to detect the autonomous behavior of mice in a new environment and explore their behavior and mental tension. It can reflect the anxiety and exploratory behavior of mice. The open field test consists of three parts: an open field reaction box and an automatic data acquisition and processing system. The background noise in the laboratory is controlled below 65 dB, and the room temperature is around 20°C. The experiment is carried out in a quiet environment. Place the animal in the center of the bottom of the box, and take pictures and time it at the same time. Clean the inner wall and bottom of the box to prevent the remaining information of the animal from affecting the next test result (such as the animal's defecation, urine, and smell). Replace the animal and continue the experiment. Observe the number of times the mouse stands and the movement trajectory during the exploration process.

[0537] 2.4 Y-maze experiment

[0538] The Y-maze alternation test was used to assess the short-term memory of animals. The order in which the mouse entered each arm and the total number of arms entered (N) were recorded. The animal's consecutive entry into three different arms was considered an alternation, and the maximum number of arm entries was defined as N-2; the spontaneous alternation rate (%) was calculated as [number of alternations / (N-2) ]×100%.

[0539] 2.5 Brain tissue sections and pathological observation

[0540] After the water maze test, 3 mice in each group were anesthetized by intraperitoneal injection of 0.3% sodium pentobarbital, the chest was opened, and saline was perfused through the apex of the heart to flush out the blood. Then 4% paraformaldehyde was used for lavage until the liver color turned white, and the head was cut off to obtain the brain tissue. Fix in 4% paraformaldehyde for more than 24 hours. Dehydration: Soak the sample tissue in a gradient of 70%-80%-95%-100% alcohol and xylene solution. Permeabilization: Treat the tissue block transparent by xylene with pure paraffin twice, each time for 1 hour. Embedding: Embed and fix the tissue block soaked in paraffin in paraffin. Sectioning: Sagittal sectioning, continuous paraffin section thickness 4μm. Baking: Paraffin sections are kept in a constant temperature baking machine at 60℃ overnight for later use.

[0541] 2.5.1 Nissl staining

[0542] Dewaxing, immerse the paraffin sections in xylene for dewaxing twice, 10 minutes each time. Alcohol gradient hydration: 100% (5min, 2 times) -95% (5min, 2 times) -90% (5min, 2 times) -85% (5min, 2 times) -75% (5min, 2 times). Wash in distilled water for 5 minutes. Stain with 1% toluidine blue at a constant temperature of 50℃ for 20min, differentiate with 70% ethanol for several minutes, and differentiate with 95% ethanol for several minutes. Randomly select 5 slices from each group for pathological observation, observe the general morphology of neurons, and calculate the average number of neurons in the dorsal cell band of hippocampal DG by the automatic image analysis system.

[0543] 2.5.2 Hematoxylin-eosin staining

[0544] (1) Brain removal: Fix and dehydrate the whole brain tissue (same steps as above). (2) Frozen sections: When the hippocampal tissue appears, adjust the thickness to 10 µm for continuous sectioning. (3) Hematoxylin staining: Attach the brain slice to a slide, stain with hematoxylin staining solution at room temperature for 5 min, and rinse with tap water. (4) Differentiation: Treat with differentiation solution for 30 s, observe the degree of differentiation under a microscope, and rinse with tap water. (5) Eosin staining: Stain with eosin staining solution at room temperature for 1 min, and rinse with tap water. (6) Dehydration and transparency: Dehydrate the slide in 70%, 85%, 95% and 100% ethanol solutions for 5 min in turn, and transparentize with xylene for 5 min. (7) Sealing: Place a drop of neutral resin next to the brain slice, seal with a coverslip, and observe under a microscope.

[0545] 2.5.3 TUNEL assay

[0546] Add anti-fluorescence quencher, cover with coverslip and avoid light, and observe the test results under a 400× optical microscope. Among them, green TUNEL represents apoptotic cells and DAPI represents in situ cell nuclei.

[0547] 2.6 Enzyme-linked immunosorbent assay

[0548] Since the accumulation of amyloid plaques is one of the key pathological signs of AD, an enzyme-linked immunosorbent assay was used to observe whether MSC-sIVs treatment affects Aβ deposition in mouse brain tissue. 48 hours after the last behavioral experiment, the animals were euthanized, and brain tissue was obtained. After PBS homogenization, it was placed in a centrifuge and centrifuged at a speed of 4000r / min at 4°C. After obtaining the homogenate, it was stored in a refrigerator at -80°C for subsequent testing. Elisa detection was performed according to the instructions of the kit.

[0549] 2.7 Determination of GSH content, SOD activity and MDA content

[0550] Oxidative stress plays an interactive role in the pathogenesis of AD. Decreased GSH levels, increased MDA content, and fluctuations in SOD activity are associated with the onset of Aβ plaques and Alzheimer's disease. To explore whether MSC-sIVs have an effect on GSH, MDA, and T-SOD, we quantitatively analyzed the GSH, MDA levels, and T-SOD activity in the cerebral cortex of 5xFAD mice. After the behavioral experiment, the mice were killed, and their brain tissues were immediately taken. The residual blood was rinsed with cold saline, dried with filter paper, weighed, cut into pieces, and added with 9 times ice saline to make a 10% aqueous tissue homogenate. The mixture was centrifuged (3500 r / min) for 10 min, and the supernatant was placed at 4°C for testing. The determination of GSH content, SOD activity, and MDA content was strictly performed according to the instructions of the kit.

[0551] 3. Statistical processing

[0552] The experimental data are expressed as mean ± standard deviation ( ). All experimental data were tested for normality. All quantitative data were analyzed using SPSS22.0. One-way ANOVA was used for variance analysis, and the least significant difference (LSD) analysis was used for post hoc test. For non-normally distributed data and data with unequal variance, nonparametric tests were used, and P values ​​< 0.05 were considered statistically significant.

[0553] 4. Experimental results

[0554] 4.1 MSC-sIVs improve spatial learning impairment in 5xFAD mice

[0555] The results of the Morris water maze test showed that the escape latency of all test groups gradually decreased. In the navigation experiment (finding the platform), the escape latency of the PBS group mice from the 4th to the 6th day was significantly higher than that of the normal group, while the escape latency of the 5xFAD mice treated with MSC-sIVs and MSC-sEVs on the 4th to 6th day was significantly lower than that of the 5xFAD mice treated with PBS. In addition, the latency of the MSC-sIVs group to reach the platform was significantly shorter than that of the PBS group on the 4th and 6th day of navigation, and the difference was statistically significant ( Fig.51 A). At the navigation stage on day 6, we found that PBS mice had difficulty finding the platform and swam around the edge compared with normal, MSC-sIVs, and MSC-sEVs groups, indicating that PBS group mice had more severe spatial memory impairment ( Fig.51 B). In the spatial exploration experiment, compared with the normal group (about 5 platform crossings and about 10.746s stay in the target quadrant), the PBS group crossed the platform significantly less (about 2.47 times) and stayed in the target quadrant for a shorter time (about 8.63s). When 5xFAD mice were treated with MSC-sIVs and MSC-sEVs, the number of platform crossings and the percentage of time spent in the target quadrant were improved, showing good learning ability. In particular, the number of platform crossings (about 3.9 times) of mice in the MSC-sIVs group was higher than that of the PBS group and almost the same as that of the normal group. At the same time, the time spent in the target quadrant (about 13.19s) was higher than that of both the PBS group and the normal group. These data indicate that 5xFAD mice have neurological damage, and MSC-sIVs and MSC-sEVs intervention can alleviate this damage to varying degrees. In particular, MSC-sIVs can significantly improve the spatial learning impairment of 5xFAD mice and alleviate their memory deficits.

[0556] 4.2 MSC-sIVs improve spontaneous exploratory behavior and tension in 5xFAD mice

[0557] Two months after the experimental treatment, the open field test was performed to detect the autonomous behavior, exploratory behavior, and tension of 5xFAD mice in the new environment. The activity trajectories of mice in the PBS group tended to be in the peripheral area of ​​the open field, while the activity trajectories of mice in the normal, MSC-sIVs, and MSC-sEVs groups tended to be in the central area, especially in the MSC-sIVs group ( Fig.52 ). Compared with the PBS group, the number of standing up in the normal group, MSC-sIVs group, and MSC-sEVs group increased significantly, indicating that they had stronger spontaneous exploratory behavior and better cognitive function. In particular, the number of standing up in the MSC-sIVs group (about 64.3 times) increased significantly, significantly higher than the PBS group (about 36 times), and almost the same as the normal group (about 65 times), indicating that MSC-sIVs can significantly stimulate the autonomous exploratory behavior of 5xFAD mice.

[0558] 4.3 MSC-sIVs improve learning and memory dysfunction in 5xFAD mice

[0559] The representative trajectory results of the Y-maze are as follows: Fig.53 As shown. The results of calculating the spontaneous alternation rate showed that the spontaneous alternation rate of mice in the PBS group (about 22%) was significantly lower than that in the normal group (about 30%) (P < 0.05); compared with the mice in the PBS group, the spontaneous alternation rate of MSC-sIVs and MSC-sEVs mice increased significantly, indicating that the learning and memory dysfunction of these mice was alleviated after treatment. In particular, the percentage of spontaneous alternation in the MSC-sIVs group (about 34%) was significantly higher than that in the PBS group (P < 0.01), and it also showed an upward trend compared with the normal group.

[0560] 4.4 MSC-sIVs alleviate oxidative stress in 5xFAD mice

[0561] Oxidative stress plays an interactive role in the pathogenesis of AD. Decreased GSH levels, elevated MDA content, and fluctuations in SOD activity are associated with the aggregation of Aβ plaques and AD. To investigate whether MSC-sIVs have an effect on GSH, MDA, and T-SOD, we quantitatively analyzed GSH, MDA levels, and T-SOD activity in the cerebral cortex of 5xFAD mice.

[0562] The brain GSH level of 5xFAD mice in the PBS group (about 52 μmol / gprot) was significantly lower than that in the normal group (about 62 μmol / gprot) (P < 0.05), indicating that the mice in the PBS group had higher levels of free radicals and cell damage, and the GSH level of 5xFAD mice after treatment with MSC-sIVs and MSC-sIVs showed an upward trend. The MDA content of mice in the PBS group (about 12.69 μmol / mg) was significantly higher than that in the normal group (about 7.2 μmol / mg) (P < 0.05), indicating that the 5xFAD mice treated with PBS had higher levels of free radicals and cell damage, and the MDA level of 5xFAD mice after treatment with MSC-sIVs and MSC-sIVs decreased significantly, and the difference was statistically significant (P < 0.05). The SOD activity of mice in the PBS group (about 0.23U / μg) was significantly lower than that in the normal group (0.39U / μg) (P<0.05), indicating that 5xFAD mice treated with PBS had higher levels of free radicals and cell damage, and the SOD activity of 5xFAD mice treated with MSC-sIVs and MSC-sIVs was significantly increased, with statistically significant differences (P<0.05). In particular, the GSH level (about 59.5μmol / gprot) and SOD activity (about 0.39U / μg) of 5xFAD mice treated with MSC-sIVs were significantly higher than those in the PBS group and comparable to those in the normal group, and the MDA content (6.5μmol / mg) was significantly lower than that in the PBS group (12.69μmol / mg).

[0563] 4.5 MSC-sIVs reduce Aβ levels in 5xFAD mice

[0564] The experimental results showed that the amyloid content observed in 5xFAD mice in the PBS group (about 19.2pg / mg) was significantly higher than that in the normal group (about 12.3pg / mg) (P < 0.05), and the amyloid content observed in 5xFAD mice after treatment with MSC-sIVs and MSC-sIVs was significantly reduced, with statistically significant differences (P < 0.05). In particular, MSC-sIVs was able to significantly reduce the Aβ plaque burden of 5xFAD mice (about 11pg / mg).

[0565] 5. Summary

[0566] After treatment with MSC-sIVs and MSC-sEVs, the level of Aβ amyloid protein in the Alzheimer's disease mouse model was significantly reduced, the oxidative stress response was effectively alleviated, neuronal cell apoptosis was inhibited, and the activation of microglia and astrocytes was weakened. More significant differences were observed in the positioning and cruising phase of the water maze behavioral experiment: compared with the PBS group, the escape latency of mice in the MSC-sIVs treatment group was significantly shortened on the 4th and 6th days of the experiment, and the reduction in the escape latency of mice in the MSC-sIVs treatment group was greater than that in the MSC-sEVs treatment group. This result suggests that MSC-sIVs may have a more significant effect in improving the spatial learning and memory ability of Alzheimer's disease mice.

[0567] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0568] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0569] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. Use of intracellular nanovesicles derived from mesenchymal stem cells in the preparation of drugs for preventing and / or treating nervous system diseases; The vesicles are prepared by a method comprising the following steps: (1) Dispersing mesenchymal stem cells in a suspension solvent and subjecting them to ultrasonic treatment; (2) centrifuging the liquid obtained in step (1) once or multiple times, discarding cell membranes and organelle fragments, and taking the supernatant; (3) subjecting the supernatant obtained in step (2) to ultracentrifugation to obtain the precipitate as intracellular nanovesicles; The amplitude of the ultrasonic treatment in step (1) is 20%-25%; the time of the ultrasonic treatment is 10-20s; The number of centrifugal treatments in step (2) is two times, and the respective parameters are: 1000-3000g, 5-20 minutes; 10000-30000g, 20-40 minutes; The ultracentrifugation parameters in step (3) are 100000-180000 g, 50-100 minutes; The nervous system disease is selected from the group consisting of optic nerve disease, cerebrovascular disease, traumatic brain injury disease and nervous system degenerative disease.

2. The use according to claim 1, characterized in that The amplitude of the ultrasonic treatment in step (1) is 20%; and / or the time of the ultrasonic treatment is 15 seconds, with a running time of 2 seconds and a pause time of 2 seconds.

3. The use according to claim 1, characterized in that The mesenchymal stem cells are selected from the group consisting of umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, dental pulp mesenchymal stem cells, placenta and amniotic fluid, and amniotic membrane mesenchymal stem cells.

4. The use according to claim 1, characterized in that The average particle size of the vesicles is 50-100 nm.

5. The use according to any one of claims 1 to 4, characterized in that: The nervous system disease is an optic nerve disease.

6. The use according to claim 5, characterized in that The optic nerve disease is optic nerve damage.

7. The use according to any one of claims 1 to 4, characterized in that: The nervous system disease is ischemic cerebrovascular disease.

8. The use according to claim 7, characterized in that The ischemic cerebrovascular disease is cerebral infarction.

9. The use according to any one of claims 1 to 4, characterized in that: The nervous system disease is a nervous system degenerative disease.

10. The use according to claim 9, characterized in that The neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

11. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug is selected from: oral dosage form, injection dosage form, skin dosage form, eye dosage form, and nasal dosage form.

12. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug is a subcutaneous administration dosage form.

13. The use according to any one of claims 1 to 4, characterized in that: The dosage form of the drug is intravitreal injection, nasal administration, intravenous injection or intraperitoneal injection.

14. The use according to any one of claims 1 to 4, characterized in that: The medicine is in the form of a cream, foam, ointment, liquid solution, gel or spray.

15. The use according to any one of claims 1 to 4, characterized in that: The medicine is an ointment.

16. The use according to any one of claims 1 to 4, characterized in that: The medicine is an emulsion, eye drops, injection, suspension or microemulsion.

17. The use according to any one of claims 1 to 4, characterized in that: The medicine is powder injection.

18. The use according to any one of claims 1 to 4, characterized in that: The drug is formulated into an eye patch or contact lens formulation.

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