Neural repair protein composition as well as preparation method and application thereof

Abstract

The invention relates to a neural repair protein composition which is prepared by the following steps: adding any one or a combination of 20-35U / mL nuclease or all-potent nuclease into a cell protein extract, carrying out enzymolysis for 15-40min under the condition of 37 + / -1 DEG C, and separating and purifying the prepared enzymatic hydrolysate, thereby obtaining the neural repair protein composition. The nerve repair protein composition disclosed by the invention has the effects of repairing cells, repairing nerve injury and the like, and can be used for repairing nerve injury caused by diseases such as central nervous injury, neurodegenerative diseases, cerebral apoplexy, cerebral trauma, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, senile dementia and the like or complications thereof; the composition has the advantages of good stability, high bioavailability, safety, effectiveness, convenience in production, storage and transportation and the like.

Description

[0001] This application is a divisional application of invention patent application No. 202310042914.3 filed on January 28, 2023. Technical Field

[0002] The present invention belongs to the field of biomedicine technology, and specifically relates to a nerve repair protein composition, a preparation method thereof and an application thereof. Background Art

[0003] Cells are the basic units of life activities and the foundation of body health. When the body's redox balance is disrupted, it will cause the interruption of redox signals and control, trigger oxidative stress damage and cause a variety of diseases. Brain ischemia and hypoxia (such as stroke, cerebral hemorrhage, long-term high-altitude work, internal carotid artery and vertebral artery occlusion and / or stenosis, etc.) and oxidative stress damage can cause a variety of neurodegenerative diseases caused by the loss of neurons or glial cells, including memory loss, Alzheimer's disease, Parkinson's disease, Alzheimer's disease, multiple sclerosis, ataxia, Huntington's disease, amyotrophic lateral sclerosis, etc. At present, there are more than 3 million Parkinson's patients, more than 200,000 ALS patients, and more than 10 million Alzheimer's patients in my country, and with the increasing aging of the global population, there are characteristics such as high incidence. Timely repair of damaged cells can significantly improve and treat related lesions.

[0004] Stroke (stenosis or occlusion of the cerebral blood supply artery resulting in blood not flowing into the brain) and cerebral hemorrhage (sudden rupture of a blood vessel in the brain) are common central nervous system diseases. They are caused by ischemic and hypoxic damage to brain tissue due to insufficient blood supply to the brain, and manifest as corresponding neurological deficit symptoms, including balance problems, hemiplegia, loss of sensation and vibration sensation, numbness, weakened reflexes, ptosis, visual field defects, aphasia and apraxia, etc. Excessive free radicals are the main cause of the aggravation of ischemia-reperfusion injury in early acute ischemic stroke. The lesion has the characteristics of high incidence, poor prognosis, easy recurrence, high disability rate and high mortality rate, which seriously affects human health and quality of life. Ataxia is mostly caused by clumsy and uncoordinated movements due to cerebellar, proprioceptive and vestibular dysfunction. When the trunk, limbs and throat muscles are involved, it can cause body balance, posture, gait and speech disorders, including cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia, etc. The etiology is complex and often a comprehensive manifestation of multiple diseases, including acute brain swelling caused by infarction, edema or hemorrhage. Brain trauma is mostly caused by external objects in the head, often causing varying degrees of permanent functional impairment, including abnormal focal symptoms in the movement, sensation, speech, vision, hearing and other regions. Peripheral nerve injury is a common clinical disease mainly caused by trauma, tumors and metabolic diseases (such as diabetes and its complications). It often leads to partial or complete loss of movement, sensation and autonomic function of the affected segment of the body, and even intractable neuralgia, which seriously affects the patient's quality of life. The "Global Diabetes Report" released by WHO in 2017 shows that the number of diabetes patients in the world is increasing year by year. There are 129 million diabetes patients in my country. Their various complications (including neuralgia and nerve damage caused by ischemia and hypoxia of peripheral nerves and peripheral nerves) lead to serious consequences such as disability and death in diabetic patients.

[0005] The clinical treatment of stroke emphasizes brain nerve protection while thrombolysis in order to reverse neuronal apoptosis and improve nerve damage. Butylphthalide can reduce intracellular calcium ion concentration, inhibit glutamate release and reduce arachidonic acid production, scavenge oxygen free radicals, increase oxidase activity, etc., acting on multiple pathological links of cerebral ischemic lesions. It is clinically used to improve local circulation of cerebral infarction lesions, reduce infarction area, reduce brain tissue damage, restore neurological function, and improve neurological deficits in patients with acute cerebral ischemic stroke. It is mainly used to treat mild and moderate acute ischemic stroke. Idarubicin and dextroborneol are antioxidants and free radical scavengers that can scavenge a variety of free radicals, reduce brain edema and tissue damage, inhibit the expression of inflammatory factors caused by cerebral ischemia-reperfusion, and reduce cell apoptosis and cell necrosis. It is clinically used to improve neurological symptoms, daily living activities and functional disorders caused by acute cerebral infarction.

[0006] When cells and microorganisms are subjected to external stimuli and exogenous stressors (including cold, heat, acid, alkali, electric current, radiation, chemicals, etc.), they will induce stress proteins in cells and microorganisms due to stress response. Document 1 (Newlimonophyllines AC from the stem of Atalantia monophylla and cytotoxicityagainst cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41: 431–437) discloses that compounds 1-16 extracted from Rutaceae plants (Atalantia monophylla) have activities such as inhibiting tumor cell growth.

[0007] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, and urine. They have the characteristics of wide sources, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and convenient collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), and interferon (IFN). They participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, and immune maturation processes. They can be used for immune regulation, tissue repair, and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome. However, MSCs products need to be refrigerated in their production, storage, transportation, and application, and their cell viability is maintained at ≤12h, which limits their therapeutic application. To this end, it is necessary to develop safe and effective nerve repair drugs to meet clinical needs. Summary of the invention

[0008] The object of the present invention is to provide a nerve repair protein composition, the preparation of which comprises the following steps:

[0009] (1) adding 20 U / mL to 35 U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract of the present invention, placing the extract at 37°C ± 1°C for enzymatic hydrolysis for 15 min to 40 min to obtain an enzymatic hydrolyzate;

[0010] (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution fraction at an ultraviolet wavelength of 280 nm is monitored and collected to obtain a method wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0011] In the preferred technical solution of the present invention, the preparation of the protein extract comprises the following steps:

[0012] S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal stem cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 2h-6h, and then separated, washed and collected, wherein the stressor is selected from any one or a combination of compounds 1-16,

[0013]

[0014]

[0015] S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof;

[0016] S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

[0017] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid (18AA) 0.02-0.05% and 3-8μmol / L of stressor.

[0018] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F1245%, RPMI164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid (18AA) 0.05% and 4-6μmol / L of stressor.

[0019] In the preferred technical solution of the present invention, the density of mesenchymal stem cells in step S-1 is 8.0×10 6 -2.0×10 7 / mL, preferably 8.0×10 6 -1.0×10 7 Pieces / mL.

[0020] In a preferred technical solution of the present invention, the mesenchymal stem cells in step S-1 are cultured in the culture medium for 3h-5h, preferably 3.5h-4.5h.

[0021] In a preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of cell washing times is 2-5 times, preferably 3-4 times.

[0022] In the preferred technical scheme of the present invention, the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm*5-10min.

[0023] In the preferred technical solution of the present invention, the ultrasonic conditions of step S-2 are: working for 3s and then resting for 1s at 2°C-8°C, 25kHZ, 360W, and ultrasonic treatment for 1-5min.

[0024] In the preferred technical solution of the present invention, the separation described in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multi-stage centrifugation, multi-stage filtration or a combination thereof, preferably 3000-7000rpm*15-25min.

[0025] In the preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min respectively.

[0026] In the preferred technical solution of the present invention, the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

[0027] In the preferred technical scheme of the present invention, 25U / mL-30U / mL of any one or a combination of nuclease or universal nuclease is added to the cell protein extract prepared by S-3, and the extract is placed at 37°C±1°C for enzymatic hydrolysis for 20min-30min to obtain an enzymatic hydrolyzate.

[0028] In a preferred technical solution of the present invention, the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

[0029] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 or the protein composition obtained in step (2) is frozen, preferably at -40°C to -20°C.

[0030] In a preferred technical scheme of the present invention, a lyophilization protectant is added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), and lyophilized to obtain a cell protein extract lyophilized preparation or a protein composition lyophilized preparation, wherein the lyophilization protectant is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

[0031] In a preferred technical solution of the present invention, the freeze-dried preparation contains 0.5-8%, preferably 1-5%, of freeze-protectant by mass percentage.

[0032] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cell protein extract obtained in step S-3 or the protein composition obtained in step (2), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

[0033] According to a preferred technical solution of the present invention, the pH of the cell protein extract freeze-dried preparation or the protein composition freeze-dried preparation is 6-8, preferably pH 7-7.5.

[0034] In a preferred technical solution of the present invention, the molecular weight of the nerve repair protein composition is 20 kDa-250 kDa, preferably 35 kDa-200 kDa.

[0035] The preferred technical solution of the present invention is that the protein composition of the nerve repair protein composition is as follows Figure 2 shown.

[0036] In a preferred technical solution of the present invention, the lyophilized preparation is reconstituted with physiological saline or 5% glucose solution before use, and then used by intravenous injection, intrathecal injection, lumbar puncture or any one or a combination thereof.

[0037] In a preferred technical solution of the present invention, the culture of the mesenchymal stem cells or the culture of the primary mesenchymal stem cells adopts a culture method in the art.

[0038] In the preferred technical solution of the present invention, the culture of mesenchymal stem cells comprises the following steps: primary mesenchymal stem cells are cultured at an initial density of 5.0×10 5 -5.0×10 6 The culture medium is added with 100 μg / ml of DMEM / F12 medium, and then cultured at 37.0°C±0.5°C and 5%±1.0% CO2 for 10-15 days. Every 2-3 days, the culture medium is observed to turn yellow and replaced by half. The culture medium contains 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin.

[0039] In the preferred technical solution of the present invention, the culture of primary mesenchymal stem cells comprises the following steps:

[0040] 1) After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2, and the medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells;

[0041] 2) Shake and collect the lower layer cells, wash with PBS, add 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, pipette gently to blow, centrifuge at 1200-1500rpm / min*5-8min, and collect the cells.

[0042] The object of the present invention is to provide a method for preparing a cell protein extract having a nerve repair effect, comprising the following steps:

[0043] S-1: The density is 5.0×10 6 / mL-5.0×10 7 Mesenchymal stem cells of 100 μg / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0°C±0.5°C, 5%±1.0% CO2 for 2h-6h, and then separated, washed and collected, wherein the stressor is selected from any one or a combination of compounds 1-16;

[0044] S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof;

[0045] S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

[0046] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid (18AA) 0.02-0.05% and 3-8μmol / L of stressor.

[0047] In the preferred technical scheme of the present invention, the culture medium of step S-1 contains DMEM / F1245%, RPMI164045%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug / mL, fibroblast growth factor (FGF) 10ug / mL, insulin transferrin 10ug / mL, compound amino acid (18AA) 0.05% and 4-6μmol / L of stressor.

[0048] In the preferred technical solution of the present invention, the density of mesenchymal stem cells in step S-1 is 8.0×10 6 -2.0×10 7 / mL, preferably 8.0×10 6 -1.0×10 7 Pieces / mL.

[0049] In a preferred technical solution of the present invention, the mesenchymal stem cells in step S-1 are cultured in the culture medium for 3h-5h, preferably 3.5h-4.5h.

[0050] In a preferred technical solution of the present invention, the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof, and the number of cell washing times is 2-5 times, preferably 3-4 times.

[0051] In the preferred technical scheme of the present invention, the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm*5-10min.

[0052] In the preferred technical solution of the present invention, the ultrasonic conditions of step S-2 are: working for 3s and then resting for 1s at 2°C-8°C, 25kHZ, 360W, and ultrasonic treatment for 1-5min.

[0053] In the preferred technical solution of the present invention, the separation in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multi-stage centrifugation, multi-stage filtration or a combination thereof, preferably 3000-7000rpm*15-25min.

[0054] In the preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min respectively.

[0055] In the preferred technical solution of the present invention, the pore size of the filter membrane for multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.

[0056] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is frozen, preferably at -40°C to -20°C.

[0057] In a preferred technical solution of the present invention, the cell protein extract obtained in step S-3 is hydrolyzed by either nuclease or omnipotent nuclease and then separated and purified.

[0058] In a preferred technical solution of the present invention, the culture of the mesenchymal stem cells or the culture of the primary mesenchymal stem cells adopts a culture method in the art.

[0059] In the preferred technical solution of the present invention, the culture of mesenchymal stem cells comprises the following steps: primary mesenchymal stem cells are cultured at an initial density of 5.0×10 5 -5.0×10 6 The culture medium is added with 100 μg / ml of DMEM / F12 medium, and then cultured at 37.0°C±0.5°C and 5%±1.0% CO2 for 10-15 days. Every 2-3 days, the culture medium is observed to turn yellow and replaced by half. The culture medium contains 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin.

[0060] In the preferred technical solution of the present invention, the culture of primary mesenchymal stem cells comprises the following steps:

[0061] 1) After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected, and placed in DMEM / F12 medium containing 10% fetal bovine serum FBS, 100ug / ml penicillin, and 100ug / ml streptomycin, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2, and the medium was replaced by half every 2-3 days until the tissue blocks crawled out of the cells;

[0062] 2) Shake and collect the lower layer cells, wash with PBS, add 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, pipette gently to blow, centrifuge at 1200-1500rpm / min*5-8min, and collect the cells.

[0063] The object of the present invention is to provide a method for preparing a nerve repair protein composition, comprising the following steps:

[0064] (1) adding 20 U / mL to 35 U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract of the present invention, placing the extract at 37°C ± 1°C for enzymatic hydrolysis for 15 min to 40 min to obtain an enzymatic hydrolyzate;

[0065] (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution fraction at an ultraviolet wavelength of 280 nm is monitored and collected to obtain a method wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0066] In a preferred technical solution of the present invention, the nuclease is selected from any one of RNA nuclease and DNA nuclease or a combination thereof.

[0067] In a preferred technical solution of the present invention, 25U / mL-30U / mL of any one or a combination of nuclease or universal nuclease is added to the cell protein extract of the present invention, and the extract is placed at 37°C±1°C for enzymatic hydrolysis for 20min-30min to obtain an enzymatic hydrolyzate.

[0068] In a preferred technical solution of the present invention, the molecular weight of the nerve repair protein composition is 20 kDa-250 kDa, preferably 35 kDa-200 kDa.

[0069] In a preferred technical solution of the present invention, the protein composition obtained in step (2) is frozen, preferably at -40°C to -20°C.

[0070] In a preferred technical scheme of the present invention, a lyophilization protective agent is added to the protein composition collected in step (2), and lyophilized to obtain a lyophilized preparation of the protein composition, wherein the lyophilization protective agent is selected from any one of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylin (HES), polyethylene glycol, ethylene glycol, phosphate, acetate, citrate, sorbitol, starch, or a combination thereof.

[0071] In a preferred technical solution of the present invention, the freeze-dried preparation contains 0.5-8%, preferably 1-5%, of freeze-protectant by mass percentage.

[0072] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the protein composition collected in step (2), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.

[0073] According to a preferred technical solution of the present invention, the lyophilized preparation has a pH of 6-8, preferably a pH of 7-7.5.

[0074] The preferred technical solution of the present invention is that the protein composition of the nerve repair protein composition is as follows Figure 2 shown.

[0075] In a preferred technical solution of the present invention, the lyophilized preparation is reconstituted with physiological saline or 5% glucose solution before use, and then used by intravenous injection, intrathecal injection, lumbar puncture or any one or a combination thereof.

[0076] Another object of the present invention is to provide a nerve repair composition, which is composed of any one or a combination of the cell protein extract with neurological efficacy or the nerve repair protein composition of the present invention and a pharmaceutically acceptable carrier.

[0077] The dosage or type of the pharmaceutically acceptable carrier of the present invention is determined according to factors such as the physicochemical properties and content of the active ingredients in the composition, the type of preparation, the dissolution and bioavailability of the preparation, and the like.

[0078] The composition of the present invention can be in the dosage form of the art and can be prepared using the formulation technology of the art.

[0079] In a preferred technical solution of the present invention, the composition is selected from any one of a lyophilized agent, a gel, a nasal spray, a liquid dressing, an injection, a patch, an ointment, a cream, an emulsion, and a suppository.

[0080] In a preferred technical solution of the present invention, the administration method of the composition is selected from any one of intravenous injection, intrathecal injection, and lumbar puncture, or a combination thereof.

[0081] Another object of the present invention is to provide a cell protein extract having nerve cell repair efficacy, a nerve repair protein composition or a composition thereof for use in preparing any one of cell repair and nerve repair products.

[0082] In the preferred technical scheme of the present invention, the neural repair is selected from the neural damage caused by any one of the central nervous system damage, neurodegenerative diseases, stroke, brain trauma, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, senile dementia, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, peripheral nerve damage, diabetes, memory loss or its complications.

[0083] In a preferred technical solution of the present invention, the ataxia is selected from any one of cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia or their complications.

[0084] Another object of the present invention is to provide the use of compound 1-16 for stress-induced stem cells to produce functional proteins with repair efficacy.

[0085] In a preferred technical solution of the present invention, the repair is selected from any one of cell repair, hair follicle repair, joint repair, and nerve repair.

[0086] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0087] Unless otherwise specified, the identification of mesenchymal stem cells MSCs in the present invention refers to "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neuroestorative clinical application".

[0088] The present invention adopts commercially available kits to detect the levels of reactive oxygen species (IOD), superoxide dismutase SOD and malondialdehyde MDA.

[0089] Compared with the prior art, the present invention has the following beneficial effects:

[0090] 1. The present invention scientifically screens a culture medium containing a stressor to induce mesenchymal stem cells to produce functional proteins with cell repair and nerve repair effects. The obtained cell protein extract, protein composition or its composition has the effects of cell repair and nerve damage repair, and can be used to repair nerve damage caused by any of the following diseases or complications of central nervous system injury, neurodegenerative diseases, stroke, brain trauma, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, senile dementia, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, peripheral nerve injury, diabetes, memory loss, cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia, and the like, and has the advantages of high purity, good stability, high bioavailability, safety and effectiveness, and easy production, storage and transportation.

[0091] 2. The preparation method of the present invention has the advantages of simple operation, environmental protection, better cost, and suitability for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] Figure 1 The electrophoretic separation results of the nerve repair protein composition of the present invention;

[0093] Figure 2 The results of high performance liquid chromatography (HPLC) testing of the nerve repair protein composition of the present invention;

[0094] Figure 3 Study on the repairing effect of the cell protein extract of the present invention on skin damage caused by UVB irradiation;

[0095] Figure 4 Effect of the cell protein extract of the present invention on the generation of active oxygen species (IOD) in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * p < 0.05 compared with the NC (negative control) group, ** indicates p < 0.01 compared with the NC (negative control) group;

[0096] Figure 5 Effect of the cell protein extract of the present invention on the production of superoxide dismutase SOD in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * p < 0.05 compared with the NC (negative control) group, ** indicates p < 0.01 compared with the NC (negative control) group;

[0097] Figure 6 Effect of the cell protein extract of the present invention on the MDA level in skin damage caused by UVB irradiation. Mean ± SD (n = 3). ## indicates p < 0.01 compared with the blank control (BC) group; * p < 0.05 compared with the NC (negative control) group, ** indicates p < 0.01 compared with the NC (negative control) group;

[0098] Figure 7 Comparison results of weekly body weight between the treatment group and the model group in a study on the damage-repairing effect of the neurorepair protein composition of the present invention on ischemic stroke;

[0099] Figure 8 TTC staining results of the treatment group and the model group in the study of the damage and repair effect of the neurorepair protein composition of the present invention on ischemic stroke;

[0100] Fig. 9 Results of immunofluorescence staining of iba1-positive cells in the treatment group and the model group in a study of the damage-repairing effect of the neurorepair protein composition of the present invention on ischemic stroke;

[0101] Fig.10 Results of immunofluorescence staining of β3Tubulin positive cells in the treatment group and the model group in the study of the damage repair effect of the neurorepair protein composition of the present invention on ischemic stroke;

[0102] Fig.11 The neurological function (NDS) scoring results in the study of the repair effect of the neurorepair protein composition of the present invention on ischemic stroke damage. DETAILED DESCRIPTION

[0103] The details of the present invention are further explained and described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0104] 1. Culture of primary mesenchymal stem cells

[0105] The culture of primary mesenchymal stem cells includes the following steps:

[0106] 1) After cleaning and disinfecting the umbilical cord, dissect the tissue, take the Wharton's jelly tissue, and cut it into 3mm 3 The tissue blocks were centrifuged, washed, and collected. They were placed in a culture bottle, and DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin was added. They were then cultured at 37°C and 5% CO2 to promote their adhesion. After the medium turned yellow every 2-3 days, half of the medium was replaced. The culture was cultured for 10-12 days until cells were seen crawling out of the edge of the tissue block.

[0107] 2) Gently shake the tissue blocks to make them fall off, collect the tissue blocks and the lower layer cells respectively, and culture the collected tissue blocks again on the wall;

[0108] 3) After washing the collected lower layer cells with PBS, add an appropriate amount of 0.25% trypsin to digest for 2min-3min, add an equal volume of trypsin stop solution to stop digestion, gently blow the bottom of the bottle with a pipette, centrifuge at 1500rpm / min*5min, and collect the cells.

[0109] 2. Subculture of primary mesenchymal stem cells (Cultivation of mesenchymal stem cells)

[0110] Subculture of primary mesenchymal stem cells (culture of mesenchymal subcultured stem cells): primary mesenchymal stem cells were cultured at an initial density of 5.0×10 5 -5.0×10 6 Add 100U / ml of the culture medium to DMEM / F12 containing 10% FBS, 100U / ml penicillin and 100ug / ml streptomycin, and then culture it at 37.0℃±0.5℃, 5%±1.0% CO2 for 10-15 days. Replace half of the culture medium after observing it turn yellow every 2-3 days.

[0111] 3. The preparation of compounds 1-16 refers to document 1 (New limonophyllines A-C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41: 431-437).

[0112] Example 1 Preparation of cell protein extract with nerve repair effect of the present invention

[0113] The method for preparing the cell protein extract having the effect of nerve repair of the present invention comprises the following steps:

[0114] (1) Mesenchymal cells were cultured at a density of 8.0×10 6 10 μmol / mL of 5% 5-nitropropene-1-nitropropene-2-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1

[0115] (2) The cells collected in step (1) were cultured at a density of 1.0×10 7 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0116] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm*20 min, and the obtained centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn to obtain the obtained cell protein extract. The obtained cell protein extract can be frozen at -40°C to -20°C as needed.

[0117] Example 2 Preparation of the nerve repair protein composition of the present invention

[0118] The preparation of the nerve repair protein composition of the present invention comprises the following steps:

[0119] (1) Add 25 U / mL of UCF.ME UltraNuclease to the cell protein extract prepared in Example 1, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0120] (2) at 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared with an elution solvent at a concentration of 10 mg / ml, and sequentially passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.1-1 ml / min, and the elution fraction with an ultraviolet wavelength of 280 nm is monitored and collected to obtain the product, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0121] Example 3 Preparation of freeze-dried preparation of nerve repair protein composition of the present invention

[0122] Mannitol was added to the cell protein composition prepared in Example 2, stirred, mixed evenly, and then freeze-dried. The resulting freeze-dried preparation contained 2.15% mannitol (m / m).

[0123] Example 4 Preparation of cell protein extract with nerve repair effect of the present invention

[0124] The method for preparing the cell protein extract having the effect of nerve repair of the present invention comprises the following steps:

[0125] (1) Mesenchymal cells were cultured at a density of 1.0×10 7 10 μmol / mL of 5% 4-nitropropene-1-nitropropene-2-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1

[0126] (2) The cells collected in step (1) were cultured at a density of 2.0×10 7 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0127] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0128] Example 5 Preparation of the nerve repair protein composition of the present invention

[0129] The preparation of the nerve repair protein composition of the present invention comprises the following steps:

[0130] (1) Add 30 U / mL of UCF.ME UltraNuclease to the cell protein extract prepared in Example 4, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0131] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate prepared in step (1) is prepared with an elution solvent at a concentration of 10 mg / ml, and sequentially passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.5 ml / min, and the protein composition is monitored and collected at an ultraviolet wavelength of 280 nm, to obtain a protein composition, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0132] Example 6 Preparation of freeze-dried preparation of nerve repair protein composition of the present invention

[0133] Dextran was added to the cell protein composition prepared in Example 5, stirred, mixed evenly, and then freeze-dried. The resulting freeze-dried preparation contained 1% dextran (m / m).

[0134] Example 7 Preparation of cell protein extract with nerve repair effect of the present invention

[0135] The method for preparing the cell protein extract having the effect of nerve repair of the present invention comprises the following steps:

[0136] (1) Mesenchymal cells were cultured at a density of 9.0×10 610 μmol / mL of 5% 5-nitropropene-1-nitropropene-2-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1

[0137] (2) The cells collected in step (1) were cultured at a density of 2.0×10 7 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0138] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0139] Example 8 Preparation of the nerve repair protein composition of the present invention

[0140] The preparation of the nerve repair protein composition of the present invention comprises the following steps:

[0141] (1) Add 20 U / mL of UCF.ME UltraNuclease to the cell protein extract obtained in Example 7, and place it at 37° C. for 30 min to obtain an enzymatic solution;

[0142] (2) At 2°C-8°C, the enzymatic hydrolyzate obtained in step (1) is prepared with an elution solvent to a concentration of 10 mg / ml, and sequentially passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 0.1-1 ml / min, and the protein composition is monitored and collected at an ultraviolet wavelength of 280 nm, to obtain a protein composition, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0143] Example 9 Preparation of freeze-dried preparation of nerve repair protein composition of the present invention

[0144] Sorbitol was added to the cell protein composition prepared in Example 8, stirred, mixed evenly, and then freeze-dried to obtain a freeze-dried preparation containing 5% sorbitol (m / m).

[0145] Example 10 Preparation of cell protein extract with nerve repair effect of the present invention

[0146] The method for preparing the cell protein extract having the effect of nerve repair of the present invention comprises the following steps:

[0147] (1) Mesenchymal cells were cultured at a density of 1.0×10 7 10 μg / mL of 5% 4-nitropropene-1-nitropropene-2-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1-nitropropene-1

[0148] (2) The cells collected in step (1) were cultured at a density of 2.0×10 7 Cells were dispersed in normal saline and sonicated for 3s, 1s at rest, and 2min at 2-8°C, 25kHz, and 360W to obtain cell lysate.

[0149] (3) The cell lysate obtained in step (2) is centrifuged at 7000 rpm for 20 min, and the resulting centrifuge is filtered through 0.45 um and 0.22 um filter membranes in turn.

[0150] Embodiment 11 Preparation of the nerve repair protein composition of the present invention

[0151] The preparation of the nerve repair protein composition of the present invention comprises the following steps:

[0152] (1) Add 30 U / mL of UCF.ME UltraNuclease to the cell protein extract obtained in Example 10, and place the extract at 37° C. for 30 min to obtain an enzymatic solution;

[0153] (2) Under the condition of 2°C-8°C, the enzymatic hydrolyzate prepared in step (1) is prepared with an elution solvent at a concentration of 10 mg / ml, and sequentially passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6×5 mm) and a high-purity silica gel liquid chromatography preparative column (SHIMSEN Ankylo C18, 5 μm, 4.6×250 mm) at an elution flow rate of 1 ml / min, and the protein composition is monitored and collected at an ultraviolet wavelength of 280 nm, to obtain a protein composition, wherein the elution solvent is composed of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8).

[0154] Example 12 Preparation of freeze-dried preparation of nerve repair protein composition of the present invention

[0155] Mannitol was added to the cell protein composition obtained in Example 11, stirred, mixed evenly, and then freeze-dried to obtain a freeze-dried preparation containing 3% mannitol (m / m).

[0156] Example 13 Preparation of freeze-dried preparation of cell protein extract of the present invention

[0157] A required amount of mannitol was added to the cell protein extract obtained in Example 1, stirred, mixed evenly, and then freeze-dried. The resulting freeze-dried preparation contained 5% mannitol (m / m).

[0158] Embodiment 14 Molecular weight distribution detection of the nerve repair protein composition of the present invention

[0159] The molecular weight distribution of the nerve repair protein composition of the present invention is detected by standard molecular weight polyacrylamide gel electrophoresis, comprising the following steps:

[0160] 1. Select a glass plate with a thickness of 1.5 mm and place it horizontally. Spread the glue solution (15% lower glue, 4% separation glue) prepared according to Table 1 on the glass plate in sequence. Insert the comb vertically into the lower glue.

[0161] Table 1

[0162] Reagents 4% separation gel 15% lower layer glue ddH2O 6ml 3.7ml 30% acrylamide mixed solution 1.33ml 8ml Tris 2.5ml (0.5M Tris pH 6.8) 4ml (1.5M Tris pH 8.8) 10% SDS 100ul 160ul 10% APS 100ul 160ul TEMED 10ul 18ul

[0163] 2. Thaw the Marker (20KDa-245KDa) frozen at -40°C, and use PBS to prepare the Marker and the freeze-dried powder of the nerve repair protein composition of Example 3, Example 6, Example 9, and Example 12 into 10ug / ul of the test sample, add 20ul of the test sample to the loading well, and perform electrophoresis at 60-80V until clear bands appear. Adjust the voltage to 100-120V until the Marker is completely separated, place the PAGE gel in Coomassie Brilliant Blue dye solution, and stop dyeing when clear bands appear on the gel. Wash with pure water, and decolorize with 10% acetic acid solution. Stop decolorizing when the gel becomes transparent. See the results. Figure 1 , wherein strip A is embodiment 3, strip B is embodiment 6, strip C is embodiment 9, and strip D is embodiment 12.

[0164] Embodiment 15 High performance liquid chromatography detection of the nerve repair protein composition of the present invention

[0165] The lyophilized powder of the nerve repair protein composition of Example 3 was dissolved in deionized water to prepare a 10 mg / ml test solution.

[0166] Chromatographic column: SHIMSEN Ankylo (300mm*4.6mm.D.,3um; P / N:380-01215-05)

[0167] Mobile phase: 50mmol / L phosphate buffer (pH=6.8), containing 300mmol / L sodium chloride; flow rate: 0.3mL / min; injection volume: 10ul; column temperature: 25℃; detection wavelength: 280nm; isocratic elution, collection time: 30min. Results are shown in Figure 2 .

[0168] Test Example 1 Study on the repairing effect of the cell protein extract of the present invention on UVB radiation damaged skin

[0169] 3D Skin Model: Commercially available from EpiKutis.

[0170] Model culture medium: DMEM basal medium.

[0171] VE working solution: take 0.5 g of VE stock solution and dissolve it in 10 mL of anhydrous ethanol to prepare a 5% stock solution; take 100 μL of 5% stock solution and dissolve it in 10 mL of model culture medium to prepare a 0.05% VE working solution.

[0172] Add 0.9 mL of model culture medium to a 6-well plate, transfer the 3D skin model to the 6-well plate, and mark the test number.

[0173] Blank control (BC): The skin model was not treated and was placed in a CO2 incubator (37°C, 5% CO2) for 48 h;

[0174] Negative control (NC): irradiation dose on the skin model surface is 600mJ / cm 2 After UVB, it was placed in a CO2 incubator (37°C, 5% CO2) for 24 h; 25 μL of 0.2% SLS working solution was added and it was placed in a CO2 incubator (37°C, 5% CO2) for 24 h;

[0175] Positive control (PC): irradiation dose on the skin model surface is 600mJ / cm 2 After UVB, place it in a CO2 incubator (37°C, 5% CO2) for 24 hours; add 25ul of 0.05% VE working solution, and place it in a CO2 incubator (37°C, 5% CO2) for 24 hours;

[0176] Experimental group: The irradiation dose on the skin model surface was 600mJ / cm 2 After UVB, it was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours; 25ul of 1% cell protein extract solution (the cell protein extract lyophilized powder obtained in Example 13 was made into a 1% solution with physiological saline), and then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0177] After incubation for 48 hours, the skin model was washed with PBS solution to remove the residual liquid inside and outside. After fixation with 4% paraformaldehyde for 24 hours, the model was removed by circumcision and observed after H&E staining. The results are shown in Figure 3 . The levels of reactive oxygen species (ROS), superoxide dismutase, and malondialdehyde (MDA) in skin damaged by UVB radiation were detected. The results are shown in Figure 4-Figure 6 The cell protein extract of the present invention has an obvious repairing effect on skin damaged by UVB radiation.

[0178] Test Example 2 Investigation of the repairing effect of the neural repair protein composition of the present invention on ischemic stroke damage

[0179] 75 SPF male SD rats aged 6-7 weeks were selected. After the experimental animals were adaptively fed with ordinary feed for 1 week, all animals underwent cerebral ischemia reperfusion surgery to establish a stroke model. After the experimental animals were reperfused, they were awakened from anesthesia and neurological function scores were immediately performed.

[0180] The experimental rats were fasted overnight the day before the experiment. After anesthesia, they were fixed on the surgical board in the supine position. A longitudinal incision of about 3 cm was made on the neck to expose and bluntly free the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). A slipknot was tied at the proximal end of the common carotid artery (CCA) to temporarily block the blood supply. A double knot was tied at the external carotid artery (ECA), and a small opening was made in the middle of the double knot with microscissors to insert the thread plug. The external carotid artery (ECA) was cut from the middle of the double knot, and the direction of the thread plug was adjusted to enter the internal carotid artery (ICA). The bifurcation of the internal carotid artery (ICA) and the external carotid artery (ECA) was used as the reference position. The thread plug was inserted to the preset scale. Resistance was felt, indicating that the head of the thread plug had reached the middle cerebral artery (MCA). The thread plug was tied and fixed. After 60 minutes of ischemia, the thread plug was pulled out and the slipknot of the common carotid artery was loosened.

[0181] 62 experimental animals survived after surgery. According to the scoring results, the experimental animals were divided into a model group (30) and a treatment group (32), and all were injected into the lateral ventricle three times, including immediate administration after cerebral ischemia reperfusion, administration 1 week after surgery, and administration 2 weeks after surgery. The treatment group (the freeze-dried powder of the neural repair protein composition of Example 3 was dissolved in normal saline, the dosage was 6.77 mgf / kg, and the administration volume was 20ul), and the model group was given the same volume of normal saline in the corresponding lateral ventricle. Three days after surgery, 2 animals were selected from each group for TTC staining; 4 weeks after surgery, 15 animals were sampled in the model group, 16 animals were sampled in the treatment group, and 1 experimental animal was selected from each group for TTC staining; 8 weeks after surgery, 13 animals were sampled in the model group, 14 animals were sampled in the treatment group, and 1 experimental animal was selected from each group for TTC staining.

[0182] During the test, the test animals were observed once a week, including death, mental state, behavioral activity, morbidity, respiration, secretions, feces, diet and drinking water. The weight of all test animals was tested once a week, and the results are shown in Figure 7 During the experiment, there was no difference in body weight between the treatment group and the model group.

[0183] The consistency of the stroke model was evaluated 3 days, 4 weeks, and 8 weeks after surgery. After blood collection, a catheter was immediately inserted into the left ventricle, and the patient was rapidly perfused with saline for 2 minutes. The brain was removed by decapitation and stained with TTC. The brain was cut into 5-7 coronal sections from front to back with a thickness of about 1.8 mm. After staining with 1% TTC at 37°C for 10 minutes, the slices were fixed with tissue fixative, scanned with a scanner, and analyzed with analysis software. It reacts with dehydrogenase in normal tissue and appears red, while the dehydrogenase activity in ischemic tissue decreases and cannot react, so there is no change and it appears pale. The results are shown in Figure 8 .

[0184] The animal limb grasping force tester was used to test and evaluate the muscle strength of the animals before and one week after surgery. The rotating rod fatigue instrument was used to test the whole body endurance of the experimental animals before and one week after surgery: the speed was 12r / min, and the experiment was carried out three times in a row, each time for 3 minutes, including the speed at the time of falling, the running duration experiment, and the three average values ​​of the distance traveled. The results are shown in Table 2. The forelimb grasping force, speed before falling, distance traveled, and duration of the treatment group were higher than those of the model group one week after surgery (P<0.01).

[0185] Table 2

[0186]

[0187]

[0188] Note: Compared with the model group, * indicates P < 0.05, ** indicates p < 0.01.

[0189] Water maze test Animal learning and memory function and activity evaluation at 4 weeks and 8 weeks after surgery: The first four days were for acquisition training experiments, and the fifth day was for platform exploration experiments. Acquisition training experiment method: First, the platform was exposed to the water surface so that the rats could see the platform. Then the rats were placed in the swimming pool. If the rats swam directly to the platform without difficulty, it meant that their swimming ability and vision were normal, and the experiment could be started. The platform was installed 5 mm below the water surface in the second quadrant. The rats were placed in the water with their backs to the pool wall. The placement position was randomly selected without repetition from one of the starting positions of the four quadrants 1, 2, 3, and 4. Each of the four quadrants was placed once, for a total of 4 times, and the time it took for the rats to find the underwater platform (escape latency) was recorded. If the escape latency exceeded 60 s, the rats were guided to the platform, allowed to stay on the platform for 10 s, and then the rats were removed and wiped dry. If necessary, the rats were baked under a 150 W incandescent lamp for 5 min and returned to the cage. Platform exploration experiment method: After the acquisition training, the platform in the second quadrant was removed on the second day, and the rats were placed in the water with their heads facing the pool wall at the starting position in the fourth quadrant. After the experiment, the number of platform crossings in the acquisition experiment was analyzed, and the results are shown in Table 3. The number of platform crossings in the treatment group at 4 weeks and 8 weeks after surgery was higher than that in the model group (P<0.01).

[0190] Table 3

[0191]

[0192] Note: Compared with the model group, * indicates P < 0.05, ** indicates p < 0.01.

[0193] Pathological examination: Tissue samples were collected with a thickness of 3 mm, dehydrated with gradient alcohol at 70%, 80%, 95%, and 100% for 30 min each, two bottles of xylene for 20 min each, and two cylinders of paraffin for 30 min each, embedded, sliced ​​to 4 microns, baked, and stained. The results are shown in Tables 4 and 5. The percentage of atrophy and calcification area of ​​the healthy side relative to the injured side in the treatment group 8 weeks after surgery were significantly lower than those in the model group (P<0.01).

[0194] Table 4

[0195]

[0196] Note: Compared with the model group, * indicates P < 0.05, ** indicates p < 0.01.

[0197] Table 5

[0198]

[0199] Note: Compared with the model group, * indicates P < 0.05, ** indicates p < 0.01.

[0200] Immunofluorescence staining results (see Table 6, Figure 9-10 ). The area of ​​iba1-positive cells in the treatment group was significantly lower than that in the model group at 4 and 8 weeks after surgery (p<0.05); the number of β3Tubulin-positive cells in the treatment group was significantly higher than that in the model group at 8 weeks after surgery (p<0.01).

[0201] Table 6

[0202]

[0203] Note: Compared with the model group, * indicates P < 0.05, ** indicates p < 0.01.

[0204] Test Example 3 Study on the damage-repairing effect of the neural repair protein composition of the present invention on ischemic stroke

[0205] 80 SPF SD male rats weighing 220-240 g were selected and the experimental animals were allowed to eat and drink freely.

[0206] The experimental animals were anesthetized with isoflurane gas and maintained. They were fixed in the supine position, and the skin was cut along the midline of the neck to expose the right common carotid artery. The nerves and fascia around the bifurcation of the common carotid artery to the base of the skull were carefully separated. The occipital artery, superior thyroid artery, lingual artery and maxillary artery of the external carotid artery were separated, ligated and cut. #The nylon thread (0.25 mm in diameter) was introduced into the internal carotid artery and inserted into the middle cerebral artery at the Willis circle to effectively block the middle cerebral artery. The length of the inserted nylon thread was 18 to 20 mm from the bifurcation of the common carotid artery. 2 hours after MCAO, the nylon thread in the lumen of the internal carotid artery was carefully withdrawn to reperfuse the internal carotid artery. Neurological function scores (NDS, see Table 7) were performed after the animals were fully awake. The modeling was successful when the experimental animal scored > 8 points.

[0207] Table 7 Neurological function scoring criteria

[0208]

[0209]

[0210] 60 rats with successful modeling were selected and randomly divided into 6 groups according to the scores, with 10 rats in each group. The drug administration method is shown in Table 8). The experimental animals were administered intravenously after reperfusion.

[0211] Table 8

[0212]

[0213]

[0214] The neurological function of the experimental animals was scored in a blinded manner before administration and 1 day, 3 days, and 5 days after administration. The experimental data were expressed as mean ± standard deviation (Mean ± SEM) and statistically analyzed using SPSS25.0 software. One-way ANOVA was performed when the variance was equal, and LSD test was used for pairwise comparison when there was a significant difference; Kruskal-Wallis nonparametric test was performed for unequal variance, and pairwise comparison was performed. The results are shown in Fig.11 Compared with before administration, the experimental group 1 showed a comparable effect on repairing stroke nerve damage as the positive control 1 and positive control 2 at 5 days after administration (P<0.05); compared with before administration, the experimental group 2 showed a significant effect on repairing nerve damage at 3 days after administration (P<0.05); compared with before administration, the experimental group 2 had a more significant effect on repairing stroke nerve damage than the positive control 1 and positive control 2 at 5 days after administration (P<0.01).

[0215] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A nerve repair protein composition, the preparation of which comprises the following steps: (1) adding 20 U / mL to 35 U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract, placing the extract under 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic solution; (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution fraction at an ultraviolet wavelength of 280 nm is monitored and collected to obtain: The elution solvent consisted of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8); The preparation of the protein extract comprises the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal stem cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 2h-6h, and then separated, washed and collected, wherein the stressor is selected from any one of compounds 1-16 or a combination thereof, S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

2. The protein composition as claimed in claim 1, wherein the culture medium in step S-1 contains DMEM / F12 42-45%, RPMI1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5-10ug / mL, fibroblast growth factor (FGF) 5-10ug / mL, insulin transferrin 5-10ug / mL, compound amino acid (18AA) 0.02-0.05% and 3-8μmol / L of stressor.

3. The protein composition according to any one of claims 1 to 2, wherein the culture medium in step S-1 contains 45% DMEM / F12, 45% RPMI 1640, 0.5% bovine serum albumin (BSA), 10 ug / mL epidermal growth factor (EGF), 10 ug / mL fibroblast growth factor (FGF), 10 ug / mL insulin transferrin, 0.05% compound amino acid (18AA) and 4-6 μmol / L stressor. 4 . The protein composition according to claim 1 , wherein the molecular weight of the nerve repair protein composition is 20 kDa-250 kDa, preferably 35 kDa-200 kDa.

5. A method for preparing a nerve repair protein composition, comprising the following steps: (1) adding 20 U / mL to 35 U / mL of any one or combination of nuclease or universal nuclease to the cell protein extract, placing the extract under 37°C ± 1°C for 15 min to 40 min to prepare an enzymatic solution; (2) at 2°C-8°C, the enzymatic hydrolysate obtained in step (1) is prepared with an elution solvent to a concentration of 5-15 mg / ml, and then passed through a chromatographic column at an elution flow rate of 0.1-1 ml / min, and the elution fraction at an ultraviolet wavelength of 280 nm is monitored and collected to obtain: The elution solvent consisted of 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8); The preparation of the protein extract comprises the following steps: S-1: The density is 5.0×10 6 / mL-1.0×10 7 Mesenchymal stem cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 2h-6h, and then separated, washed and collected, wherein the stressor is selected from any one of compounds 1-16 or a combination thereof, S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

6. A cell protein extract having a nerve repairing effect, comprising the following steps: S-1: The density is 5.0×10 6 / mL-5.0×10 7 Mesenchymal stem cells of 100 μg / mL were placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor. Then, the cells were cultured at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 2 h to 6 h, and then separated, washed, and collected. The stressor is selected from any one or a combination of compounds 1-16; S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 pcs / mL is dispersed in a solvent, and then subjected to ultrasonic treatment at 2°C-8°C to obtain a cell lysate, wherein the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

7. A method for preparing a cell protein extract having a nerve repairing effect, comprising the following steps: S-1: The density is 5.0×10 6 / mL-5.0×10 7 Mesenchymal stem cells of 100 cells / mL are placed in a culture medium containing DMEM / F12 40-50%, RPMI 1640 40-50%, bovine serum albumin (BSA) 0.1-2%, epidermal growth factor (EGF) 1-15ug / mL, fibroblast growth factor (FGF) 1-15ug / mL, insulin transferrin 1-15ug / mL, compound amino acid (18AA) 0.01-0.1% and 2-10μmol / L stressor, and then cultured at 37.0℃±0.5℃, 5%±1.0% CO2 for 2h-6h, and then separated, washed and collected, wherein, The stressor is selected from any one or a combination of compounds 1-16; S-2: Collect cells at a density of 5.0×10 6 / mL-5.0×10 7 The cells were dispersed in a solvent and then subjected to ultrasonic treatment at 2°C-8°C to obtain cell lysis products. The solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof; S-3: After the cell lysate obtained in step S-2 is separated, the obtained separation solution is filtered through 0.45um and 0.22um filter membranes in sequence.

8. A nerve repair composition, comprising the nerve repair protein composition according to claims 1-4 or the cell protein extract according to claim 6 and a pharmaceutically acceptable carrier.

9. Use of the nerve repair protein composition according to claims 1 to 4, the cell protein extract according to claim 6, or the nerve repair composition according to claim 8 in preparing any product for cell repair or nerve repair.

10. Use of compound 1-16 for stress-induced stem cells to produce functional proteins with repair effects.

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