Method for treating syringomyelia by using MSC-derived exosome

Abstract

The invention provides an application of an exosome in preparation of a medicine for treating syringomyelia. The MSCs-sourced exosome is used for treating syringomyelitis for the first time, and the MSCs-sourced exosome is low in immunogenicity, high in safety and more stable. Therefore, the traditional Chinese medicine composition is easily accepted by patients and is low in cost. And secondly, the exosome is small in size, so that the exosome is conveyed in an intrathecal injection manner, and is not easy to block. Many researches show that the exosome from the MSCs has the effects of protecting nerves and regulating immune microenvironment, can play the same curative effect as the MSCs, has no proliferation ability, no tumorigenicity or immunological rejection risk, and has higher safety than the direct transplantation of the MSCs, so that the exosome better meets the requirements of clinical transformation ethics. Intrathecal exosome injection is expected to become an ideal administration way for treatment of spinal cord diseases such as syringomyelia.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mesenchymal stem cells, and particularly relates to a method for treating syringomyelia by using MSC-derived exosomes. BACKGROUND

[0002] Syringomyelia (SM) is a chronic and progressive spinal cord degenerative disease characterized by the formation of abnormal cavities filled with fluid in the spinal cord, often causing pain, paresthesia or sensory dissociation, limb weakness and other motor or / and sensory dysfunction. The progression of the spinal cord cavity leads to spinal cord atrophy, softening, demyelination, axonal damage and decreased neurons. When the cavity involves the gray matter area around the central canal, the anterior commissure area spinothalamic tract and the posterior horn sensory neurons are damaged first, causing patients to have sensory dissociation. As the disease progresses, patients may further develop motor dysfunction such as muscle weakness and muscle atrophy, which severely affects the quality of life. Epidemiological studies have shown that more than 50% of patients with syringomyelia are at risk of severe nerve damage and progressive disability.

[0003] The pathophysiological mechanism of the spinal cord cavity is mainly related to the abnormal cerebrospinal fluid dynamics, which is often caused by subarachnoid obstruction due to various reasons (such as Chiari malformation, spinal cord tumor, trauma, arachnoiditis, etc.). Current clinical treatment mainly relies on surgical decompression (such as occipital foramen decompression, subarachnoid shunt, spinal cord cavity peritoneal shunt, etc.) to restore the balance of cerebrospinal fluid dynamics. Although surgical intervention can temporarily slow down or stop the progression of the cavity, 20-50% of patients still have persistent or recurrent neurological symptoms after surgery. This is mainly because surgical intervention can only relieve mechanical compression and restore cerebrospinal fluid circulation, but cannot reverse the already formed nerve function damage. Simply improving cerebrospinal fluid circulation is difficult to achieve the reconstruction of damaged nerve function. Drug therapy has limited effect and cannot be cured. Cell therapy can inhibit inflammation and reduce the size of the cavity, but it faces the risk of immune rejection, low cell survival rate and tumorigenesis. In addition, due to its low survival rate and large amount of cells required, cell therapy limits its application to single injection or multiple injections within a short period of time, which cannot maintain its long-term effect in the body. Therefore, exosome therapy can well fill this gap.

[0004] Exosomes are a class of extracellular vesicles with a diameter of about 30-150 nm, which play many functions as shuttles between cells, transporting various substances such as proteins, RNA, lipids and carbohydrates between cells. As a treatment method, exosomes are less affected by the inflammatory microenvironment of the damaged site; higher safety, lower cost. Compared with cells, exosomes can also be relatively easily and safely genetically manipulated to promote efficacy. Finally, due to their small size, exosomes delivered by intravenous injection are less likely to cause vascular obstruction. Compared with transplanting viable and replicating cell populations, exosomes have many advantages such as immune compatibility, reducing the risk of tumor formation, spreading infection, cell rejection, penetrating the blood-brain barrier, and avoiding potential pulmonary embolism. Exosomes are more stable and easier to store. Therefore, exosomes have great potential and are very suitable for recovery after syringomyelia. In recent years, research has found that MSCs exosomes exhibit unique potential in the treatment of nervous system diseases. In various nervous system disease models such as spinal cord injury, stroke, and neurodegenerative diseases, MSCs exosomes have been shown to have multiple neuroprotective and neurorestorative effects such as promoting nerve regeneration, inhibiting inflammatory response, reducing cell apoptosis, regulating angiogenesis and scar formation, and exerting a therapeutic effect similar to "cell-free therapy", avoiding the risk of MSC transplantation and immune risk, and meeting the clinical translation ethical requirements.

[0005] At present, there are very few studies on the use of MSCs to treat syringomyelia. In 2022, a case report claimed that MSCs could effectively treat syringomyelia, but the study only had one patient, which may not be very convincing. Another study was reported in a rat syringomyelia model, but the transplanted cells survived in the animal body for a short time and may not be able to function for a long time. At present, there is no related report on the application of MSCs exosomes intrathecal injection to treat syringomyelia. Therefore, at present, the method of developing and applying exosome therapy to treat syringomyelia should be systematically developed. SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a method for treating syringomyelia by using MSC-derived exosomes, which provides a new method for treating syringomyelia.

[0007] In one aspect of the present application, the use of exosomes in the preparation of a drug for treating syringomyelia is provided, wherein the exosomes have at least one of the following effects:

[0008] (1) promoting the proliferation and migration ability of spinal cord neural stem cells;

[0009] (2) inhibiting the apoptosis of spinal cord neural stem cells;

[0010] (3) promoting the reduction of the volume and length of the syringomyelia;

[0011] (4) improving sensory dysfunction, preferably enhancing pain and temperature sensation sensitivity; more preferably reducing mechanical pain withdrawal threshold and shortening thermal pain withdrawal latency;

[0012] (5) inhibiting apoptosis in the spinal cord cavity, preferably inhibiting neural cell apoptosis, more preferably inhibiting neuron and oligodendrocyte apoptosis;

[0013] (6) promoting the expression of neurotrophic factors and anti-inflammatory factors, and improving the neural regeneration microenvironment; preferably promoting the expression of BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor);

[0014] (7) promoting myelination and axonal regeneration.

[0015] In an embodiment of the present application, the exosomes are derived from stem cells.

[0016] In an embodiment of the present application, the stem cells are selected from mesenchymal stem cells.

[0017] In an embodiment of the present application, the exosomes have a particle size of 30-150 nm, preferably 50-150 nm, and an average particle size of about 121 nm.

[0018] In an embodiment of the present application, the exosomes are separated and purified by ultracentrifugation, density gradient centrifugation, ultrafiltration or chromatography. Preferably, the exosomes are separated and purified from MSC culture supernatant by ultracentrifugation.

[0019] In an embodiment of the present application, the exosomes are further modified.

[0020] In an embodiment of the present application, the modification methods include but are not limited to: (a) surface modification of antibodies, aptamers or small molecule ligands to enhance targeting; (b) loading of nerve regeneration related nucleic acids, proteins or small molecule drugs.

[0021] In a second aspect of the present application, a composition is provided for use in the preparation of a medicament for treating syringomyelia, wherein the composition comprises the above-mentioned exosomes.

[0022] In an embodiment of the present application, the composition has at least one of the following effects:

[0023] (1) promoting the proliferation and migration of spinal cord neural stem cells;

[0024] (2) inhibiting apoptosis of spinal cord neural stem cells;

[0025] (3) promoting the reduction of spinal cord cavity volume and length;

[0026] (4) improving sensory dysfunction, preferably enhancing pain and temperature sensation, more preferably reducing mechanical pain withdrawal threshold and shortening thermal pain withdrawal latency;

[0027] (5) inhibiting apoptosis in the spinal cord cavity, preferably inhibiting neural cell apoptosis, more preferably inhibiting neuron and oligodendrocyte apoptosis;

[0028] (6) promoting the expression of neurotrophic factors and anti-inflammatory factors, and improving the neural regeneration microenvironment; preferably promoting the expression of BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor);

[0029] (7) promoting myelination and axonal regeneration.

[0030] In an embodiment of the present application, the composition further comprises a pharmaceutically acceptable carrier.

[0031] In an embodiment of the present application, the carrier is selected from a buffer, physiological saline, a gel, a liposome or a polymer nanoparticle.

[0032] In an embodiment of the present application, the composition is formulated as an injection, a sustained-release preparation, a microsphere preparation or an implant.

[0033] In an embodiment of the present application, the injection is an intravenous injection, an intrathecal injection, a ventricular injection or a local injection.

[0034] In an embodiment of the present application, the intrathecal administration comprises a single bolus injection, continuous injection or delivery by an implanted pump.

[0035] In an embodiment of the present application, the composition is administered by continuous intrathecal injection, with a frequency of 1-2 times per week, or 1-3 times every 2 or 3 weeks, for a course of 1-4 weeks.

[0036] In an embodiment of the present application, the composition is further added with a neuroprotective agent, an anti-inflammatory drug, an anti-apoptotic factor or a drug promoting axonal regeneration when prepared.

[0037] In a third aspect of the present application, a composition comprising the above-mentioned exosome and a pharmaceutically acceptable carrier for treating syringomyelia is provided.

[0038] In an embodiment of the present application, the exosome is derived from mesenchymal stem cells.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] The application first proposes the use of MSCs-derived exosomes (MSC-Exos) to treat syringomyelia, which has low immunogenicity, high safety and is more stable. Therefore, it is easily accepted by patients and has a lower cost. Secondly, due to the small volume of exosomes, intrathecal injection is used to deliver exosomes, which is not easy to block. Many studies have shown that MSCs-derived exosomes have neuroprotective and immune microenvironment regulating effects, can exert the same efficacy as MSCs, and exosomes have no proliferation capacity, no tumorigenic or immune rejection risk, and higher safety than direct MSCs transplantation, so it is more in line with the clinical conversion ethics requirements.

[0041] Since exosomes can directly break through the BSCB restriction, intrathecal injection can target the hollow compression area and the surrounding injury area with cerebrospinal fluid circulation. In addition, intrathecal injection can avoid liver / kidney metabolic burden and systemic toxicity, and can also prolong the local action time and achieve long-acting slow release. Finally, intrathecal injection has minimally invasive and repeatability, which is more in line with the clinical treatment method and is easily accepted by patients. Therefore, intrathecal injection of exosomes is expected to become an ideal drug delivery route for the treatment of spinal cord diseases such as syringomyelia. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 The identification results of exosomes;

[0043] Figure 2 The in vitro functional verification results of MSCs exosomes;

[0044] Figure 3 The flowchart of MSCs exosome transplantation into a syringomyelia animal model;

[0045] Figure 4 The functional repair results after MSCs exosome transplantation;

[0046] Figure 5 The biological safety evaluation results;

[0047] Figure 6 The results of MSCs exosomes inhibiting apoptosis in syringomyelia;

[0048] Figure 7 The results of MSCs exosomes promoting the secretion of neurotrophic factors;

[0049] Figure 8 The results of MSCs exosomes promoting myelin formation and axon regeneration in the anterior commissure area. DETAILED DESCRIPTION

[0050] The technical solutions of the present application will be further described in detail below in conjunction with specific embodiments. It should be understood that the following examples are only illustratively described and explained, and should not be interpreted as limiting the scope of protection of the present application. Any technology implemented based on the above description of the present application is covered within the scope of the present application.

[0051] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0052] The term "exosome" generally refers to a microvesicle having a diameter of between about 30 nm to about 150 nm, and in some embodiments, the exosome has a size of about 40 nm to about 150 nm. In some embodiments, the exosome has a diameter of about 50 nm to about 150 or about 60 to about 140 nm, about 70 to about 130, about 80 to about 120, or about 90 to about 110 nm. In some embodiments, the exosome has a diameter of about 30, 35, 50, 75, 100, 110, 125, or 150 nm. In some embodiments, the exosome has an average size of about 30, about 35, about 50, about 75, about 100, about 110, about 125, or about 150 nm; or an average diameter of about about 30 to about 150, about 40 to about 150, about 50 nm to about 150, about 60 to about 140 nm, about 70 to about 130, about 80 to about 120, or about 90 to about 110 nm. At least a portion of the membrane of the exosome is directly obtained from a cell. The exosome can be detected or isolated from any suitable sample such as a body fluid.

[0053] The term "isolated" means separated from its natural environment and at least partially purified. Exosomes can be isolated from any appropriate biological sample from a mammal, including but not limited to whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced or non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige preadipocytes, etc.). In one embodiment of the present application, the exosomes are isolated from mesenchymal stem cells. Methods of isolation include centrifugation, ultracentrifugation, filtration or ultrafiltration. Exosomes can be isolated by any method known to one skilled in the art. Exemplary methods for isolating exosomes are described in the following references (Losche et al., 2004; Mesri and Altieri, 1998; Morel et al., 2004). Additionally, exosomes can also be isolated via flow cytometry as described in (Combes et al., 1997). By way of example, one acceptable protocol for isolating exosomes includes ultracentrifugation, differential centrifugation based on density and size differences, commonly used in laboratory research. The following is a prior art-based preparation method, first, collect and pre-treat the supernatant: MSCs are cultured using mesenchymal stem cell serum-free medium (NC0103), when MSCs reach 70-80% confluence, culture for 16-48 hours. Collect the supernatant (CM), transfer to a 50 mL centrifuge tube, store at 4°C or -80°C. Count the number of cells (about 1.5 x 10 6 Cells / bottle, viability > 95%) to standardize the yield. Second, remove cells and debris: centrifuge at 300-3,000 x g for 10-20 minutes. Transfer the supernatant and filter through a 0.22 μm filter membrane (to remove apoptotic bodies and residual debris). Remove microvesicles: centrifuge at 2,000-10,000 x g for 10-30 minutes. First ultracentrifugation: transfer the filtrate to an ultracentrifuge tube, ultracentrifuge at 100,000 x g for 60-90 minutes. Carefully aspirate the supernatant and retain the pellet (exosome pellet). Wash and second ultracentrifugation: resuspend the pellet with 200-500 μL of PBS. Ultracentrifuge again at 100,000 x g for 60-90 minutes. Aspirate the supernatant and resuspend the final pellet in 200-500 μL of PBS.

[0054] The "mesenchymal stem cells" can be autologous, allogeneic, or xenogeneic. The term "autologous" refers to the donor of the MSCs and the recipient of the exosomes (or isolated population of exosomes) derived from the MSCs being the same subject. The term "allogeneic" refers to the donor of the MSCs and the recipient of the exosomes (or isolated population of exosomes) derived from the MSCs being different subjects. The term "xenogeneic" refers to the donor of the MSCs and the recipient of the exosomes (or isolated population of exosomes) derived from the MSCs being subjects of different species. In a particular embodiment, the MSCs from which the exosomes are derived are allogeneic. The MSCs can be derived from any animal, preferably a mammal, including non-primate (e.g., a cow, a pig, a horse, a cat, a dog, a rat, or a mouse) and a primate (e.g., a monkey, or a human). In a particular embodiment, the MSCs are human MSCs. In a particular embodiment, the MSCs can be derived from adipose or from bone marrow.

[0055] A "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, stabilizers, gels, binders, excipients, disintegrants, lubricants, and combinations thereof, as known to those of ordinary skill in the art (Remington's, 1990). The use of any of these conventional carriers will be contemplated in therapeutic or pharmaceutical compositions unless their incompatibility with the active ingredient is shown. The compositions used in the present application can include different types of carriers, depending on whether the composition is administered in solid, liquid, or aerosol form.

[0056] In an embodiment of the present application, the composition comprising exosomes can be administered intravenously, intracerebrally, intracranially, intrathecally to the gray matter region using any method known to those of ordinary skill in the art. For intrathecal use, injection solutions or pump depot formulations can be used. In certain embodiments, the pharmaceutical composition comprises about 2% to about 75% of exosomes by weight of the composition or about 25% to about 60% of exosomes by weight of the composition. The dosage administered (both quantity and dose) depends on the subject being treated, the condition of the subject, the protection desired, and the route of administration. For example, the dosage of the therapeutic agent can be about 0.0001 milligrams to about 1.0 milligrams, or about 0.001 milligrams to about 0.1 milligrams, or about 0.1 milligrams to about 1.0 milligrams, or even about 10 milligrams, or the like, per dose. Multiple doses can be administered, for example, at least 100 milligrams or more.

[0057] Combination therapy: Certain embodiments of the present invention provide a therapy of administering or applying one or more second active pharmaceutical ingredients for the treatment or prevention of a disease, such as spinal cord injury. The second active pharmaceutical ingredient can be administered before, simultaneously with, or after exosome administration. Exemplarily, it may include neuroprotective agents, anti-inflammatory drugs, anti-apoptotic factors, or drugs that promote axonal regeneration. The neuroprotective agents, anti-inflammatory drugs, anti-apoptotic factors, or drugs that promote axonal regeneration involved in the present invention can be used to enhance the efficacy of exosome therapy for spinal cord injury. Neuroprotective agents include methylprednisolone (which reduces inflammation and oxidative stress by inhibiting NF-κB and TNF-α), lelizumab (which blocks sodium channels and reduces excitotoxicity), resveratrol (which activates the Wnt / β-catenin pathway, providing antioxidant and anti-apoptotic effects), ginsenosides (such as Rg1, which inhibit inflammation and glial scarring), curcumin (which inhibits NF-κB and reduces edema), green tea polyphenols (such as EGCG, which regulate mitochondrial function), anthocyanins (which enhance NRF2 and provide antioxidant effects), apigenin (which reduces IL-1β and TNF-α), cannabidiol (which inhibits caspase-3), vitamins C / D / E (which reduce ROS and NLRP3 activation), melatonin (which scavenges ROS), estradiol-17β (which supports neuronal survival), creatine, N-acetylcysteine, active protein C, ginkgo extract (EGb761, which inhibits cPLA2), diperoxyvanadate (which activates Akt / mTOR), and annexin A1 (which inhibits caspase-3). Anti-inflammatory drugs include methylprednisolone, nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, indomethacin, and naproxen, which inhibit Rho-A, minocycline (suppresses immune response), chitosan (reduces TNF-α), erythropoietin, estrogen, etanercept (anti-TNF-α), rolipram (PDE4 inhibitor), anti-α4β1 integrin, substance P, and G-CSF. Anti-apoptotic factors include resveratrol, EGCG, anthocyanins, cannabidiol, vitamins C / E, melatonin, GDNF (activates Akt / PI3K), annexin A1, disperoxide vanadate, and miR-21 mimics (inhibit PTEN). Drugs promoting axonal regeneration include resveratrol, epoetin B (promotes axonal elongation), retinoic acid (enhances neural differentiation), GDNF, disperoxide vanadate, methylprednisolone, and NSAIDs. These drugs work synergistically with exosomes to improve functional recovery in syringomyelia through mechanisms such as anti-inflammation, anti-oxidation, anti-apoptosis, or promotion of nerve regeneration.

[0058] The terms "intrathecal injection" or "intrathecal administration" refer to the route of drug administration via injection into the spinal canal, more specifically into the subarachnoid space, so that it reaches the cerebrospinal fluid (CSF). Intrathecal injection can include lumbar puncture, intracardiac (including intraventricular (ICV)), suboccipital / cisternal, and / or C1-2 puncture. For example, exosomes can be introduced via lumbar puncture to diffuse throughout the subarachnoid space.

[0059] The terms "patient" or "subject" refer to a living mammalian organism such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or subject is a primate. Non-limiting examples of human patients include adults, adolescents, infants, and fetuses.

[0060] "Treatment" includes: (1) suppressing disease in a subject or patient experiencing or exhibiting disease pathology or symptomology (e.g., suppressing further development of pathology and / or symptomology), (2) improving disease in a subject or patient experiencing or exhibiting disease pathology or symptomology (e.g., reversing pathology and / or symptomology), and / or (3) achieving disease relief in a subject or patient experiencing or exhibiting disease pathology or symptomology.

[0061] The term "effective" means sufficient to achieve the desired, expected result. "Effective amount," "therapeutic effective amount," or "pharmaceutical effective amount," when used in the context of treating a patient or subject with a compound, refers to the amount of the compound that, when administered to a subject or patient for the treatment or prevention of a disease, is sufficient to achieve the stated treatment or prevention of that disease.

[0062] Example 1

[0063] Exosome preparation and identification

[0064] Exosomes were isolated and purified from MSC culture supernatant using ultracentrifugation and systematically identified. Transmission electron microscopy showed that the isolates exhibited a typical saucer-like shape with a clear and intact membrane structure. They were uniform in morphology and distribution at different magnifications, with no obvious aggregation observed. Figure 1 A). Nanoparticle tracking analysis (NTA) results showed that the vesicle particle size was mainly distributed in the 50-150 nm range, exhibiting a unimodal normal distribution, with an average particle size of approximately 121 nm. Figure 1 B). Western blot analysis confirmed that the isolates were enriched in the expression of exosome characteristic markers HSP70, TSG101, and CD9, while the endoplasmic reticulum marker Calnexin was negatively expressed, ruling out organelle contamination. Figure 1 C). To facilitate subsequent tracking studies, we performed a Dil labeling experiment. The results showed that the exosomes exhibited a clear red fluorescent signal, indicating that the exosomes were successfully labeled with the Dil dye. Figure 1 (D) This provides a basis for subsequent exosome tracing experiments. These characteristics fully meet the exosome identification standards set by the International Society for Extracellular Vesicles (ISEV), confirming the successful acquisition of high-purity exosomes.

[0065] Example 2

[0066] In vitro functional verification of MSCs exosomes

[0067] To evaluate the biological function of MSC-Exos, primary rat spinal cord neural stem cells (SCNSCs) were chosen as the main research object. The effects of MSC-Exos on the proliferation, migration and apoptosis inhibition of SCNSCs were evaluated to predict the potential of MSC-Exos in promoting endogenous repair of the spinal cord.

[0068] SCNSCs were isolated and cultured from E13-16 day SD fetal rats and identified. Under light microscopy, the cells showed typical suspended neurosphere structure Figure 2 A). Immunofluorescence staining showed that these cells highly expressed neural stem cell markers Nestin, ependymal cell marker Foxj1, and neural stem cell markers BLBP and Sox2, and the co-expression of Ki67 confirmed that these cells had good proliferation activity Figure 2 B). These results confirmed that we successfully obtained SCNSCs.

[0069] In terms of proliferation, after co-culturing SCNSCs with MSC-Exos for 24 hours, it was found by EdU detection that the proportion of EdU positive cells (proliferating cells) was significantly higher than that of the control group Figure 2 C, 2D). It was proved that MSC-Exos could significantly promote the proliferation of SCNSCs. In terms of migration, the scratch test results showed that SCNSCs added with MSC-Exos showed stronger migration ability at 24h and 48h Figure 2 E, 2F), indicating that MSC-Exos treatment could effectively promote the migration of SCNSCs.

[0070] Considering the key influence of apoptosis in the repair of central nervous system injury, we explored the role of MSC-Exos in inhibiting apoptosis. By adding H2O2 to the SCNSCs culture medium to induce SCNSCs apoptosis, and then adding MSC-Exos to the experimental group for 24 hours, and only adding SCNSCs culture medium to the control group. The TUNEL staining results showed that compared with the control group, the number of TUNEL positive cells in the exosome treatment group was significantly reduced Figure 2 G), quantitative analysis confirmed the significant apoptosis inhibition effect of MSC-Exos Figure 2 H).

[0071] In summary, in vitro experiments showed that MSC-Exos could enhance the proliferation and migration ability of spinal cord neural stem cells, and had a significant apoptosis inhibition effect. This provides an experimental basis for the application of MSC-Exos in the in vivo repair of spinal cord cavities.

[0072] Example 3

[0073] In vivo experiments of MSCs exosomes

[0074] 1. Animal model of MSCs exosome transplantation into spinal cord cavitation

[0075] This study was designed with four groups: the Sham group (sham-operated group, normal control), the SM group (syringomyelia group, disease control), the DS+Vehicle group (decompression surgery + control group, treatment control), and the DS+Exo group (decompression surgery + exosome treatment group, treatment group). Experimental procedure: At week 0, syringomyelia modeling or sham surgery was performed. At week 4, rats meeting the criteria for syringomyelia were screened by MRI and underwent decompression surgery and intrathecal injection of MSC-Exos (DS+Exo group: 150 μg / 20 μL exosomes; DS+Vehicle group: equal volume of saline); the Sham and SM groups underwent only sham decompression surgery. At weeks 5 and 7, the DS+Exo and DS+Vehicle groups received supplemental intrathecal injections at the same dosage. Two weeks after treatment (i.e., week 6 of the experiment), samples were taken from some rats to assess changes in the microenvironment; MRI examinations were performed at four (eight) and eight (twelfth) weeks after treatment to assess the mid- to long-term effects of the treatment. Final sample analysis was conducted at eight weeks after treatment as the experimental endpoint. Figure 3 ).

[0076] 2. Functional restoration after MSCs exosome transplantation

[0077] To objectively evaluate the impact of MSC-Exos treatment on syringomyelia, we used T2-weighted MRI sequences to acquire sagittal and axial images of the spinal cord, and used 3D reconstruction technology to visually demonstrate the dynamic changes in the morphology of syringomyelia in each group. Figure 4 A). Quantitative analysis showed that at the experimental endpoint (12 weeks): the average cavity volume in the SM group increased significantly by 45.29% compared to 4 weeks, and the average cavity length increased by 4.94%, indicating that untreated cavities showed a progressive development trend; the average cavity volume in the DS+Vehicle group decreased by 12.89% compared to 4 weeks, and the average length decreased by 5.10%, but neither reached statistical significance, suggesting that simple decompression surgery has limited effectiveness; the average cavity volume and length in the DS+Exo group decreased by 51.11% and 36.77% respectively, and the differences were statistically significant. Figure 4 (B, 4C). These imaging data objectively confirm that MSC-Exos treatment can significantly promote the reduction of cavity volume and length.

[0078] Clinically, patients with syringomyelia often exhibit dissociated sensory and motor dysfunction, and similar functional abnormalities can be observed in animal models. To assess the treatment effect, behavioral tests were performed on rats in each group every 2 weeks starting from week 0, including the Von Frey test (pain sensation), hot plate test (temperature sensation), and foot slip test (motor coordination).

[0079] In terms of sensory function, Von Frey test and hot plate test were used to evaluate the changes of pain and temperature sensation, respectively. The results showed that the paw withdrawal threshold and pain behavior latency of rats in three groups except the Sham group were significantly increased within 4 weeks after modeling, indicating that the formation of spinal cord cavity led to a significant decrease in pain and temperature sensation. After treatment intervention, the sensory function of DS+Vehicle and DS+Exo groups began to gradually recover, while the SM group remained at a high level or continued to worsen. In terms of mechanical pain sensation, the pain threshold of DS+Vehicle and DS+Exo groups was significantly lower than that of SM group from the 8th week, but the improvement effect of DS+Exo group was more significant (D); in terms of temperature sensation, the latency of two treatment groups was significantly shorter than that of SM group from the 8th week, and the temperature sensation of DS+Exo group was significantly better than that of DS+Vehicle group at 12 weeks (E). In the foot error experiment, the score of DS+Exo group increased significantly over time, which was better than that of DS+Vehicle and SM groups (4F). At 12 weeks, the proportion of high score increased and the proportion of low score decreased in DS+Exo group, indicating that the exosome treatment group could promote the recovery of nerve function. The above results show that exosome treatment can more effectively improve the sensory dysfunction after spinal cord syringomyelia decompression. Figure 4 Figure 4 E); in the foot error experiment, the score of DS+Exo group increased significantly over time, which was better than that of DS+Vehicle and SM groups (4F). At 12 weeks, the proportion of high score increased and the proportion of low score decreased in DS+Exo group, indicating that the exosome treatment group could promote the recovery of nerve function. The above results show that exosome treatment can more effectively improve the sensory dysfunction after spinal cord syringomyelia decompression.

[0080] 3、Biological safety evaluation

[0081] To evaluate the safety of intrathecal injection of MSC-Exos, we performed systematic histological examination of the main organs of rats in the exosome group and the Sham group after 8 weeks of treatment. HE staining results showed that there were no obvious pathological changes in the lung, liver, spleen, kidney and heart tissues of rats in the DS+Exo group compared with the Sham group: the alveolar structure was complete without obvious inflammatory cell infiltration; the liver cell cords were arranged in order, and the central vein and portal area structure were normal; the white and red pulp structure of the spleen was clear, and the lymphoid follicle shape was complete; the glomerular and renal tubular morphology was normal; the myocardial fibers were arranged regularly, and the cross striations were clear (Fig. 6) (D). This indicates that intrathecal injection of MSC-Exos has no obvious toxic effect on important organs of rats and has good biological safety. Figure 5

[0082] 4、MSCs exosomes inhibit apoptosis in spinal cord cavity

[0083] ​​Following central nervous system injury, various pathological factors such as inflammation, oxidative stress, and mechanical damage can induce apoptosis. Excessive neuronal apoptosis can exacerbate neurological deficits and hinder endogenous tissue repair. Given that apoptosis triggered by the inflammatory microenvironment is a key characteristic of spinal cord injury, this invention further investigated whether a similar apoptotic mechanism exists in syringomyelia and evaluated the effect of MSC-Exos on apoptosis. By detecting the expression of Cleaved-Caspase3 (a key enzyme executing apoptosis), it was found that compared with the Sham group, the number of Cleaved-Caspase3-positive cells was significantly increased in the SM group. Figure 6 A, 6B). This result indicates that the pathological process of syringomyelia is accompanied by significant apoptosis. There was no significant difference between the DS+Vehicle group and the SM group; however, the number of Cleaved-Caspase3 positive cells in the DS+Exo group was significantly reduced compared to the DS+Vehicle group. Figure 6 A, 6B) suggest that MSCs exosomes play an anti-apoptotic role in syringomyelia.

[0084] To clarify the protective effect of MSCs-Exos on functional neurons, analysis was conducted on neurons (NeuN... + ) and oligodendrocytes (Olig2) + The expression of Cleaved-Caspase 3 in NeuN was analyzed. Results showed that in the DS+Exo group, NeuN... + With Cleaved-Caspase-3 + Co-labeled cells and Olig2 + and Cleaved-Caspase-3 + The proportion of co-labeled cells was significantly lower than that in the SM group and the DS+Vehicle group. Figure 6 A, 6C, 6D). Western blot analysis confirmed that, compared with other groups, the expression of Cleaved-Caspase-3 was significantly decreased in the DS+Exo group, and the expression of the anti-apoptotic protein Bcl-2 was significantly upregulated, while the expression of the pro-apoptotic protein Bax was significantly downregulated. Figure 6 E, 6F). The above results indicate that MSC-Exos can significantly inhibit apoptosis in the tissues surrounding syringomyelia, especially showing a significant protective effect on neurons and oligodendrocytes, creating favorable conditions for the repair of syringomyelia.

[0085] 5. MSCs exosomes promote the secretion of neurotrophic factors.

[0086] In addition to the reduction of inflammation level, the effect of MSC-Exos on the expression of neurotrophic factors in the cavity tissue was further explored. Immunofluorescence staining showed that the expression levels of BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor) in the DS+Exo group were significantly higher than those in other groups 2 weeks after treatment Figure 7 A-C). These results indicate that MSC-Exos injection can significantly promote the expression of neurotrophic factors and anti-inflammatory factors, and reshape the microenvironment conducive to nerve regeneration.

[0087] 6. MSCs exosomes promote myelin formation and axon regeneration in the anterior commissure region

[0088] The anterior commissure of the spinal cord white matter is the most vulnerable area of the spinal cord cavity, and as the crossing place of the spinothalamic tract that transmits superficial sensory information, its structural integrity is crucial for sensory function. To evaluate the repair of this area, we performed double immunofluorescence staining of MBP (myelin marker) and NF-L (axonal marker) in the anterior commissure region of each group 8 weeks after treatment Figure 8 ). The results showed that the positive density of MBP and NF-L in the SM group was significantly lower than that in the Sham group, and immunofluorescence showed that the myelin and axon in the anterior commissure region were squeezed and destroyed by the spinal cord cavity. Although the expression of MBP and NF-L increased after simple surgical decompression (DS+Vehicle group), there was no significant difference compared with the SM group; while the density of MBP and NF-L in the DS+Exo group was significantly higher than that in the DS+Vehicle group, and immunofluorescence also showed that the structure of axon and myelin was more complete. These results indicate that MSC-Exos treatment can effectively promote axon regeneration and myelin formation in the anterior commissure region.

[0089] Finally, it should be pointed out that the above examples are only used to illustrate the technical solutions of the present application and not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. Use of an exosome in the preparation of a drug for the treatment of syringomyelia.

2. The application as described in claim 1, characterized in that: The exosomes have at least one of the following functions: (1) Promotes the proliferation and migration of spinal cord neural stem cells; (2) Inhibits apoptosis of spinal cord neural stem cells; (3) Promotes the reduction of the volume and length of syringomyelia; (4) Improve sensory dysfunction, preferably by enhancing pain and temperature sensitivity; more preferably by reducing the mechanical pain withdrawal threshold and shortening the thermal pain withdrawal latency. (5) Inhibit apoptosis of cells in syringomyelia, preferably inhibiting apoptosis of nerve cells, and more preferably inhibiting apoptosis of neurons and oligodendrocytes; (6) Promote the expression of neurotrophic factors and anti-inflammatory factors, and improve the neural regeneration microenvironment; preferably promote the expression of BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor); (7) Promotes myelin formation and axon regeneration.

3. The application as described in claim 1, characterized in that: The exosomes are derived from stem cells; Preferably, the stem cells are selected from mesenchymal stem cells.

4. The application as described in claim 1, characterized in that: The exosomes have a particle size of 30-150 nm, preferably 50-150 nm, and an average particle size of about 121 nm. Preferably, the exosomes are purified by ultracentrifugation, density gradient centrifugation, ultrafiltration, or chromatography; preferably, the exosomes are obtained by ultracentrifugation from the culture supernatant of MSCs.

5. Use of a composition in the preparation of a medicament for treating syringomyelia, wherein the composition comprises exosomes as described in any one of claims 1-4.

6. The application as described in claim 5, characterized in that, The composition has at least one of the following effects: (1) Promotes the proliferation and migration of spinal cord neural stem cells; (2) Inhibits apoptosis of spinal cord neural stem cells; (3) Promotes the reduction of the volume and length of syringomyelia; (4) Improve sensory dysfunction, preferably by enhancing pain and temperature sensitivity; more preferably by reducing the mechanical pain withdrawal threshold and shortening the thermal pain withdrawal latency. (5) Inhibit apoptosis of cells in syringomyelia, preferably inhibiting apoptosis of nerve cells, and more preferably inhibiting apoptosis of neurons and oligodendrocytes; (6) Promote the expression of neurotrophic factors and anti-inflammatory factors, and improve the neural regeneration microenvironment; preferably promote the expression of BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor); (7) Promotes myelin formation and axon regeneration.

7. The application as described in claim 5, characterized in that, The composition further comprises a pharmaceutically acceptable carrier; Optionally, the carrier is selected from buffer solution, saline, gel, liposome or polymer nanoparticle.

8. The application as described in claim 5, characterized in that, The composition is formulated as an injection, a sustained-release formulation, a microsphere formulation, or an implant; Optionally, the injectable agent is an intravenous injection, an intrathecal injection, an intraventricular injection, or a local injection. Optionally, the intrathecal administration includes a single bolus injection, continuous injection, or delivery via an implantable pump; Optionally, the composition is administered via continuous intrathecal injection.

9. The application as described in claim 5, characterized in that, The composition is further supplemented with neuroprotective agents, anti-inflammatory drugs, anti-apoptotic factors, or drugs that promote axonal regeneration during preparation.

10. A composition comprising the exosomes as described in any one of claims 1-4 and a pharmaceutically acceptable carrier for the treatment of syringomyelia; Optionally, the exosomes are derived from mesenchymal stem cells.

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