WO2023143522A1 - 一种神经修复蛋白组合物及其制备方法和其应用 - Google Patents

一种神经修复蛋白组合物及其制备方法和其应用 Download PDF

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WO2023143522A1
WO2023143522A1 PCT/CN2023/073582 CN2023073582W WO2023143522A1 WO 2023143522 A1 WO2023143522 A1 WO 2023143522A1 CN 2023073582 W CN2023073582 W CN 2023073582W WO 2023143522 A1 WO2023143522 A1 WO 2023143522A1
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protein composition
cells
present
nerve
preparation
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French (fr)
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王宇
高文勇
陈琳
李建军
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北京达尔文细胞生物科技有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a nerve repair protein composition, a preparation method thereof and an application thereof.
  • Cells are the basic unit of life activities and the foundation of body health. When the body's redox balance is disrupted, redox signaling and control will be disrupted, leading to oxidative stress damage and various diseases. Cerebral ischemia and hypoxia (such as stroke, cerebral hemorrhage, long-term plateau operation, occlusion and/or stenosis of internal carotid artery and vertebral artery, etc.) The resulting neurodegenerative diseases include memory loss, Alzheimer's disease, Parkinson's disease, senile dementia, multiple sclerosis, ataxia, Huntington's disease, amyotrophic lateral sclerosis, etc.
  • Cerebral ischemia and hypoxia such as stroke, cerebral hemorrhage, long-term plateau operation, occlusion and/or stenosis of internal carotid artery and vertebral artery, etc.
  • the resulting neurodegenerative diseases include memory loss, Alzheimer's disease, Parkinson's disease, senile dementia,
  • Stroke stenosis or occlusion of cerebral blood supply arteries prevents blood from flowing into the brain
  • cerebral hemorrhage saccharinum rupture of blood vessels in the brain
  • Symptoms of corresponding neurological deficits including balance problems, hemiplegia, loss of sensation and vibration sensation, numbness, decreased reflexes, ptosis, visual field defects, aphasia, and apraxia, etc.
  • Ataxia is mostly clumsy and uncoordinated movement caused by cerebellar, proprioceptive and vestibular dysfunction.
  • Traumatic brain injury is mostly brain trauma caused by foreign objects, often causing permanent dysfunction of varying degrees, including abnormal focal symptoms in areas such as movement, sensation, speech, vision, and hearing.
  • Peripheral nerve injury is a common clinical disease mainly caused by factors such as trauma, tumors, and metabolic diseases (such as diabetes and its complications), which often leads to partial or total loss of motor, sensory, and autonomic functions of the affected segments of the body, and even stubborn Neuralgia seriously affects the quality of life of patients.
  • the World Health Organization released the "Global Diabetes Report” in 2017, showing that the number of people with diabetes worldwide is increasing year by year.
  • Butylphthalide can reduce the concentration of intracellular calcium ions, inhibit the release of glutamic acid and reduce the production of arachidonic acid, scavenge oxygen free radicals, increase the activity of oxidase, etc., and act on multiple pathological links of cerebral ischemic lesions. It is used to improve the local circulation of cerebral infarction lesions, reduce the infarct size, reduce brain tissue damage, and restore neurological function. It is used for the improvement of neurological deficits in patients with acute cerebral ischemic stroke. ischemic stroke.
  • Edaravone dexaborneol is an antioxidant and a free radical scavenger, which 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, activities of daily living and dysfunction caused by acute cerebral infarction.
  • Mesenchymal stem cells have self-replication and multi-directional differentiation potential, widely exist in bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amniotic membrane, peripheral blood, muscle, Urine and other tissues have the characteristics of wide sources, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and convenient collection.
  • SCF stem cell growth factor
  • NEF nerve growth factor
  • IL-6 interleukin-6
  • IL-7 interleukin-7
  • TNF tumor necrosis factor
  • IFN interferon
  • the object of the present invention is to provide a kind of nerve restoration protein composition, and its preparation comprises the following steps:
  • the enzymolysis solution prepared in step (1) is configured with an eluting solvent of 5-15 mg/ml, and passed through the chromatographic column at an elution flow rate of 0.1-1ml/min, Monitor and collect the elution fraction with an ultraviolet wavelength of 280nm, namely, wherein the elution solvent is composed of 300mmol/L sodium chloride in 50mmol/L phosphate buffer (pH6.8).
  • the preparation of the protein extract comprises the following steps:
  • S-1 Put the passaged mesenchymal stem cells with a density of 5.0 ⁇ 10 6 cells/mL-1.0 ⁇ 10 7 cells/mL in a 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 2-10 ⁇ mol/L stressor medium, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 for 2h-6h, separated, washed, and collected cells, among which , the stressor is selected from any one of compounds 1-16 or a combination thereof,
  • S-2 Disperse the collected cells in the solvent at a density of 5.0 ⁇ 10 6 cells/mL-5.0 ⁇ 10 7 cells/mL, and then place them under the condition of 2°C-8°C for ultrasonic treatment to prepare cell lysate , wherein, the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof;
  • PBS phosphate buffer saline
  • TBPS buffer TBST buffer
  • Tris buffer Tris buffer
  • step S-3 After separating the cell lysate prepared in step S-2, the obtained separation liquid is filtered through 0.45um and 0.22um filter membranes successively to obtain the final product.
  • 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 3-8 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • the culture medium of step S-1 contains DMEM/F12 45%, RPMI1640 45%, 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 4-6 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • EGF epidermal growth factor
  • FGF fibroblast Growth factor
  • insulin transferrin 10ug/mL
  • compound amino acid (18AA) 0.05% compound amino acid (18AA) 0.05%
  • the density of passaged mesenchymal stem cells in step S-1 is 8.0 ⁇ 10 6 -2.0 ⁇ 10 7 cells/mL, preferably 8.0 ⁇ 10 6 -1.0 ⁇ 10 7 cells/mL.
  • the passaged mesenchymal stem cells in step S-1 are cultured in the culture medium for 3h-5h, preferably 3.5h-4.5h.
  • the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer Or a combination thereof, the number of cell washings is 2-5 times, preferably 3-4 times.
  • the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation condition is 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm* 5-10min.
  • the ultrasonic conditions of step S-2 are: working at 2°C-8°C, 25kHZ, 360W for 3s, followed by an interval of 1s, ultrasonic treatment for 1-5min.
  • the separation described in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multistage centrifugation, multistage filtration or a combination thereof, preferably 3000-7000rpm*15- 25min.
  • the multistage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min in sequence.
  • the filter membrane pore size of the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.
  • any one or combination of 25U/mL-30U/mL nuclease or totipotent nuclease is added to the cell protein extract prepared in S-3, and it is placed at 37°C ⁇ Enzymolysis was carried out at 1°C for 20min-30min to obtain an enzymolysis solution.
  • the nuclease is selected from any one of RNA nuclease, DNA nuclease or a combination thereof.
  • the cell protein extract or The protein composition prepared in step (2) is stored frozen, preferably at -40°C to -20°C.
  • a lyoprotectant is added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2), and freeze-dried to obtain a cell protein extract lyophilized preparation Or protein composition lyophilized formulation, wherein, the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerin, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylycerin (HES ), polyethylene glycol, ethylene vinylene, phosphate, acetate, citrate, sorbitol, starch, any one or a combination thereof.
  • the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerin, sucrose, trehalose, glucose, lactose, maltose, dextran, tricaprylycerin (HES ), polyethylene glycol, ethylene vinylene, phosphate, acetate, citrate, sorb
  • the lyophilized preparation contains 0.5-8%, preferably 1-5%, of the lyoprotectant in terms of mass percentage.
  • a protein stabilizer is optionally added to the cell protein extract prepared in step S-3 or the protein composition prepared in step (2), wherein the protein stabilizer is selected from white Any of protein, zinc salt, and aluminum salt.
  • the pH of the lyophilized preparation of the cell protein extract or the protein composition is 6-8, preferably 7-7.5.
  • the molecular weight of the nerve repair protein composition is 20kDa-250kDa, preferably 35kDa-200kDa.
  • the protein composition in the nerve repair protein composition is shown in FIG. 2 .
  • the freeze-dried preparation is reconstituted with physiological saline or 5% glucose solution before use, and then used by any one of intravenous injection, intrathecal injection, lumbar puncture or a combination thereof.
  • the culture or primary passage of the mesenchymal stem cells are cultured using culture methods in the art.
  • the cultivation of the passaged mesenchymal stem cells includes the following steps: adding primary mesenchymal stem cells to the passage medium at an initial density of 5.0 ⁇ 10 5 -5.0 ⁇ 10 6 cells/ml culture medium at 37.0°C ⁇ 0.5°C and 5% ⁇ 1.0% CO 2 for 10-15 days, and after every 2-3 days, half of the subculture medium was replaced after the subculture medium was observed to turn yellow.
  • the subculture medium contains 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin in DMEM/F12 medium.
  • the cultivation of the primary mesenchymal stem cells comprises the following steps:
  • the tissue is dissected, and the Huatong glue layer tissue is taken, cut into small pieces of 3 mm 3 , centrifuged, cleaned, and the tissue pieces are collected, and placed in 10% fetal bovine serum FBS, 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 , half of the medium was replaced every 2-3 days, Culture until the tissue block climbs out of the cells;
  • 10% fetal bovine serum FBS 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium
  • the object of the present invention is to provide a method for preparing a cell protein extract with nerve repairing effect, comprising the following steps:
  • S-1 Put mesenchymal passaged stem cells with a density of 5.0 ⁇ 10 6 cells/mL-5.0 ⁇ 10 7 cells/mL in a 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 2-10 ⁇ mol/L stressors, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 for 2h-6h, separated, washed, and collected cells, wherein, The stressor is selected from any one of compounds 1-16 or a combination thereof;
  • S-2 Disperse the collected cells in the solvent at a density of 5.0 ⁇ 10 6 cells/mL-5.0 ⁇ 10 7 cells/mL, and then place them under the condition of 2°C-8°C for ultrasonic treatment to prepare cell lysate , wherein, the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof;
  • PBS phosphate buffer saline
  • TBPS buffer TBST buffer
  • Tris buffer Tris buffer
  • step S-3 After separating the cell lysate prepared in step S-2, the obtained separation liquid is filtered through 0.45um and 0.22um filter membranes successively to obtain the final product.
  • 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 3-8 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • the culture medium of step S-1 contains DMEM/F12 45%, RPMI1640 45%, 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 4-6 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • EGF epidermal growth factor
  • FGF fibroblast Growth factor
  • insulin transferrin 10ug/mL
  • compound amino acid (18AA) 0.05% compound amino acid (18AA) 0.05%
  • the density of passaged mesenchymal stem cells in step S-1 is 8.0 ⁇ 10 6 -2.0 ⁇ 10 7 cells/mL, preferably 8.0 ⁇ 10 6 -1.0 ⁇ 10 7 cells/mL.
  • the passaged mesenchymal stem cells in step S-1 are cultured in the culture medium for 3h-5h, preferably 3.5h-4.5h.
  • the solvent for washing cells in step S-1 is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer Or a combination thereof, the number of cell washings is 2-5 times, preferably 3-4 times.
  • the separation described in step S-1 is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation condition is 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm* 5-10min.
  • the ultrasonic conditions of step S-2 are: working at 2°C-8°C, 25kHZ, 360W for 3s, followed by an interval of 1s, ultrasonic treatment for 1-5min.
  • the separation described in step S-3 is selected from any one of 2000-8000rpm*10-30min centrifugation, multistage centrifugation, multistage filtration or a combination thereof, preferably 3000-7000rpm*15-25min .
  • the multistage centrifugation in step S-3 is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min in sequence.
  • the filter membrane pore size of the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.
  • the cell protein extract prepared in step S-3 is frozen, preferably at -40°C to -20°C.
  • the cell protein extract prepared in step S-3 is enzymatically hydrolyzed by any one of nuclease or totipotent nuclease, and then separated and purified.
  • the culture of the passaged mesenchymal stem cells or the culture of the primary mesenchymal stem cells adopts a culture method in the art.
  • the cultivation of the passaged mesenchymal stem cells includes the following steps: adding primary mesenchymal stem cells to the passage medium at an initial density of 5.0 ⁇ 10 5 -5.0 ⁇ 10 6 cells/ml culture medium at 37.0°C ⁇ 0.5°C and 5% ⁇ 1.0% CO 2 for 10-15 days, and after every 2-3 days, half of the subculture medium was replaced after the subculture medium was observed to turn yellow.
  • the subculture medium contains 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin in DMEM/F12 medium.
  • the cultivation of the primary mesenchymal stem cells comprises the following steps:
  • the tissue is dissected, and the Huatong glue layer tissue is taken, cut into small pieces of 3 mm 3 , centrifuged, cleaned, and the tissue pieces are collected, and placed in 10% fetal bovine serum FBS, 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 , half of the medium was replaced every 2-3 days, Culture until the tissue block climbs out of the cells;
  • 10% fetal bovine serum FBS 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium
  • the object of the present invention is to provide a preparation method of nerve repair protein composition, comprising the steps of:
  • the enzymolysis solution prepared in step (1) is configured with an eluting solvent of 5-15 mg/ml, and passed through the chromatographic column at an elution flow rate of 0.1-1ml/min, Monitor and collect the elution fraction with an ultraviolet wavelength of 280nm, namely, wherein the elution solvent is composed of 300mmol/L sodium chloride in 50mmol/L phosphate buffer (pH6.8).
  • the nuclease is selected from any one of RNA nuclease, DNA nuclease or a combination thereof.
  • nuclease or totipotent nuclease or any combination thereof is added to the cell protein extract of the present invention, and placed at 37°C ⁇ 1°C Enzyme hydrolyzed for 20min-30min to prepare enzymolyzate.
  • the molecular weight of the nerve repair protein composition is 20kDa-250kDa, preferably 35kDa-200kDa.
  • the protein composition obtained in step (2) is frozen, preferably at -40°C to -20°C.
  • a lyoprotectant is added to the protein composition collected in step (2), and lyophilized to obtain a lyophilized formulation of the protein composition, wherein the lyoprotectant is selected from mannitol, Sorbitol, Dextran, Glycerin, Sucrose, Trehalose, Glucose, Lactose, Maltose, Dextran, Tricaprylin (HES), Polyethylene Glycol, Ethylene Diene, Phosphate, Acetate, Citric Acid Any one or combination of salt, sorbitol, starch.
  • the lyophilized preparation contains There is 0.5-8% of lyoprotectant, preferably 1-5%.
  • 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.
  • the pH of the freeze-dried preparation is 6-8, preferably pH 7-7.5.
  • the protein composition in the nerve repair protein composition is shown in FIG. 2 .
  • the freeze-dried preparation is reconstituted with physiological saline or 5% glucose solution before use, and then used by any one of intravenous injection, intrathecal injection, lumbar puncture or a combination thereof.
  • Another object of the present invention is to provide a nerve repair composition, said composition is composed of any one of the present invention's cell protein extract or nerve repair protein composition or its combination and a pharmaceutically acceptable carrier composition.
  • the amount or type of the pharmaceutically acceptable carrier of the present invention depends on the physical and chemical properties and content of the active ingredients in the composition, the type of preparation, the dissolution and bioavailability of the preparation and other factors.
  • composition of the present invention can be a dosage form in the art, and can be prepared by using the formulation technology in the art.
  • the composition is selected from any one of freeze-dried formulations, gels, nasal sprays, liquid dressings, injections, patches, ointments, creams, emulsions, and suppositories.
  • the administration mode of the composition is selected from intravenous injection, Either intrathecal injection, lumbar puncture, or a combination thereof.
  • Another object of the present invention is to provide the application of the cell protein extract, nerve repair protein composition or its composition with nerve cell repair function in the preparation of any one of cell repair and nerve repair products.
  • the nerve restoration is selected from central nervous system injury, neurodegenerative disease, stroke, traumatic brain injury, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, senile dementia, multiple Nerve damage caused by any of sclerosis, Huntington's disease, amyotrophic lateral sclerosis, peripheral nerve damage, diabetes, memory loss or its complications.
  • the ataxia is selected from any one of cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia or their complications.
  • Another object of the present invention is to provide the application of compound 1-16 for stress-induced stem cells to produce functional proteins with repair function.
  • the repair is selected from any one of cell repair, hair follicle repair, joint repair, and nerve repair.
  • said percentage is volume/volume percentage;
  • said percentage is volume/weight percentage;
  • said percentages are weight/volume percentages; the remainder are weight/weight percentages.
  • the identification of mesenchymal stem cells MSCs of the present invention refers to "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application”.
  • the present invention uses a commercial kit to detect the levels of active oxygen (IOD), superoxide dismutase SOD and malondialdehyde MDA.
  • IOD active oxygen
  • SOD superoxide dismutase
  • MDA malondialdehyde
  • the present invention has the following beneficial effects:
  • the present invention scientifically screens the medium containing stressors to induce mesenchymal stem cells to produce functional proteins with cell repair and nerve repair effects, and the obtained cell protein extracts, protein compositions or compositions thereof have cell repair and nerve repair functions.
  • Repair nerve damage and other effects can be used to repair central nervous system damage, neurodegenerative diseases, stroke, traumatic brain injury, 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, or its concurrent Nerve damage caused by disease, and has the advantages of high purity, good stability, high bioavailability, safe and effective, and convenient for production, storage and transportation.
  • the preparation method of the present invention has the advantages of simple operation, environmental protection, better cost, and suitability for industrial production.
  • Fig. 3 cell protein extract of the present invention is to the repairing effect of skin damage caused by UVB irradiation use research
  • Fig. 4 Effect of cell protein extract of the present invention on generation of reactive oxygen species (IOD) in skin damage caused by UVB irradiation.
  • Fig. 7 The weekly body weight comparison results of the treatment group and the model group in the research on the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke;
  • Fig. 9 The results of immunofluorescent staining of iba1-positive cells in the treatment group and the model group in the research on the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke;
  • Figure 10 The results of immunofluorescent staining of ⁇ 3Tubulin positive cells in the treatment group and the model group in the research on the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke;
  • the cultivation of primary mesenchymal stem cells includes the following steps:
  • Subculture of primary mesenchymal stem cells (cultivation of passaged mesenchymal stem cells): the primary mesenchymal stem cells were added to a medium containing 10% FBS , 100 U /ml penicillin and 100ug/ml streptomycin in DMEM/F12 medium Then culture it under the conditions of 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 for 10-15 days, every 2-3 days, observe the yellowing of the medium, and replace half of the medium.
  • the preparation of compound 1-16 refers to literature 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).
  • Example 1 The preparation of the cell protein extract with nerve repair effect of the present invention
  • the passaged mesenchymal cells were added at a density of 8.0 ⁇ 10 6 cells/mL containing DMEM/F12 45%, RPMI1640 45%, 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 5 ⁇ mol/L of the compound 16 culture medium, then place it in 37 °C, 5 After culturing for 4 hours under the condition of %CO 2 , it was centrifuged at 1200 rpm for 5 minutes, washed 3 times with PBS, and the cells were collected;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 1.0 ⁇ 10 7 cells/mL, sonicate for 3s with a gap of 1s at 2-8°C, 25kHz, 360W, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • the prepared cell protein extract can be frozen and stored at -40°C to -20°C as needed.
  • Embodiment 2 Preparation of nerve repair protein composition of the present invention
  • the preparation of the nerve repair protein composition of the present invention comprises the following steps:
  • the enzymolysis solution prepared in step (1) was configured to 10mg/ml with an eluting solvent, and then passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 ⁇ 5mm ), a high-purity silica gel liquid chromatography preparative column (SHIMSENAnkylo C18, 5 ⁇ m, 4.6 ⁇ 250mm), the elution flow rate is 0.1-1ml/min, monitoring and collecting the elution fraction with an ultraviolet wavelength of 280nm, that is, wherein, the elution
  • the solvent was composed of 300mmol/L sodium chloride in 50mmol/L phosphate buffer (pH6.8).
  • 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).
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 2.0 ⁇ 10 7 cells/mL, sonicate for 3s with a gap of 1s at 2-8°C, 25kHz, 360W, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • the preparation of the nerve repair protein composition of the present invention comprises the following steps:
  • the enzymolysis solution prepared in step (1) is configured to 10mg/ml with an eluting solvent, and passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 ⁇ 5mm ), a high-purity silica gel liquid chromatography preparative column (SHIMSENAnkylo C18, 5 ⁇ m, 4.6 ⁇ 250mm), the elution flow rate is 0.5ml/min, and the protein composition with an ultraviolet wavelength of 280nm is monitored and collected, and the elution solvent It consists of 50mmol/L phosphate buffer (pH6.8) containing 300mmol/L sodium chloride.
  • the passaged mesenchymal cells were added at a density of 9.0 ⁇ 10 6 cells/mL containing DMEM/F12 45%, RPMI1640 45%, 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 5 ⁇ mol/L of the compound 14 culture medium, then place it in 37 °C, 5 After culturing for 4 hours under the condition of %CO 2 , it was centrifuged at 1200 rpm for 5 minutes, washed 3 times with PBS, and the cells were collected;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 2.0 ⁇ 10 7 cells/mL, sonicate for 3s with a gap of 1s at 2-8°C, 25kHz, 360W, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • the preparation of the nerve repair protein composition of the present invention comprises the following steps:
  • the enzymolysis solution prepared in step (1) was configured to 10mg/ml with an eluting solvent, and then passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 ⁇ 5mm ), high-purity silica gel liquid chromatography preparative column (SHIMSENAnkylo C18, 5 ⁇ m, 4.6 ⁇ 250mm), the elution flow rate is 0.1-1ml/min, monitoring and collecting the protein composition with an ultraviolet wavelength of 280nm, that is, wherein, the elution
  • the solvent was composed of 300mmol/L sodium chloride in 50mmol/L phosphate buffer (pH6.8).
  • the passaged mesenchymal cells were added at a density of 1.0 ⁇ 10 7 cells/mL containing DMEM/F12 45%, RPMI1640 45%, 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 6 ⁇ mol/L of the compound 15 culture medium, then it was placed in 37 °C, 5 After culturing for 4 hours under the condition of %CO 2 , it was centrifuged at 1200 rpm for 5 minutes, washed 3 times with PBS, and the cells were collected;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 2.0 ⁇ 10 7 cells/mL, sonicate for 3s with a gap of 1s at 2-8°C, 25kHz, 360W, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • the preparation of the nerve repair protein composition of the present invention comprises the following steps:
  • the enzymolysis solution prepared in step (1) was configured to 10mg/ml with an eluting solvent, and then passed through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 ⁇ 5mm ), a high-purity silica gel liquid chromatography preparative column (SHIMSENAnkylo C18, 5 ⁇ m, 4.6 ⁇ 250mm), the elution flow rate is 1ml/min, and the protein composition with an ultraviolet wavelength of 280nm is monitored and collected, and the elution solvent is obtained by 50mmol/L phosphate buffer (pH6.8) contains 300mmol/L sodium chloride.
  • Example 13 The preparation of the freeze-dried preparation of the cell protein extract of the present invention
  • Standard molecular weight polyacrylamide gel electrophoresis is used to detect the molecular weight distribution of the nerve repair protein composition of the present invention, comprising the following steps:
  • Example 15 High performance liquid chromatography detection of the nerve repair protein composition of the present invention
  • Example 3 After dissolving the lyophilized powder of nerve repair protein composition in Example 3 with deionized water, it was made into a 10 mg/ml test solution.
  • 3D skin model Commercially available from EpiKutis.
  • Model culture medium DMEM basal medium.
  • VE working solution absorb 0.5g VE stock solution, dissolve it in 10mL absolute ethanol, and prepare 5% mother solution; absorb 100 ⁇ L 5% mother solution, dissolve it in 10mL model culture medium, and prepare 0.05% VE working solution.
  • Blank control (BC) without any treatment, the skin model was placed in a CO 2 incubator (37°C, 5% CO 2 ) and incubated for 48 hours;
  • Negative control After irradiating the surface of the skin model with UVB at a dose of 600mJ/cm 2 , place it in a CO 2 incubator (37°C, 5% CO 2 ) and incubate it for 24h; add 25 ⁇ L of 0.2% SLS to work solution, and incubated in a CO 2 incubator (37°C, 5% CO 2 ) for 24 hours;
  • PC Positive control
  • Test group After the skin model surface was irradiated with UVB at a dose of 600mJ/cm 2 , it was placed in a CO 2 incubator (37°C, 5% CO 2 ) and incubated for 24h; 25ul of 1% cell protein extract solution was added (The lyophilized powder of cell protein extract prepared in Example 13 was made into a 1% solution with physiological saline), and then placed in a CO 2 incubator (37° C., 5% CO 2 ) for incubation for 24 hours.
  • Test Example 2 Investigation of the repairing effect of the nerve repair protein composition of the present invention on ischemic stroke damage
  • the experimental rats were fasted one night before the experiment. After anesthesia, they were fixed in the supine position on the operating board. A longitudinal incision of about 3 cm was made in the neck to expose and bluntly free the common carotid artery (CCA), external carotid artery (ECA), Internal carotid artery (ICA), tie a slipknot at the proximal end of the common carotid artery (CCA), temporarily block the blood supply, tie a double knot with the external carotid artery (ECA), and use microscissors to cut a small opening in the middle of the double knot to penetrate Thread plug, cut off the external carotid artery (ECA) from the double knot, adjust the direction of the thread plug so that it enters the internal carotid artery (ICA), and take the bifurcation of the internal carotid artery (ICA) and external carotid artery (ECA) as a reference position , the thread plug is inserted to the preset scale, and
  • the experimental animals were divided into model group (30 animals) and treatment group (32 animals), and they were injected into the lateral ventricle three times, including administration immediately after cerebral ischemia-reperfusion, administration one week after operation, Administered 2 weeks after surgery.
  • the treatment group the freeze-dried powder of the nerve repair protein composition of Example 3 was dissolved in physiological saline, the dosage was 6.77mgf/kg, and the administration volume was 20ul), and the model group was given the same volume of physiological saline corresponding to the lateral ventricle.
  • test animals were observed once a week, including death, mental state, behavioral activity, disease, breathing, secretions, feces, diet and drinking water.
  • the body weight of all experimental animals was tested once a week, and the results are shown in Figure 7.
  • the treatment group and the model group There was no difference in body weight.
  • the muscle strength of animals before operation and 1 week after operation was detected and evaluated by animal limb grip tester.
  • the whole-body endurance of the test animals before operation and 1 week after operation was detected by a rotary rod fatigue instrument: the rotating speed was 12r/min, and the experiments were carried out continuously for 3 times, each time for 3 minutes, including the average value of the falling speed, running continuous experiment, and traveling distance.
  • the results are shown in Table 2.
  • the forelimb grasping force, speed before falling, traveling distance and duration in the treatment group were higher than those in the model group one week after operation (P ⁇ 0.01).
  • Tissue samples were taken with a thickness of 3mm, dehydrated with gradient alcohol at 70%, 80%, 95%, 100% for 30 minutes each, two bottles of xylene for 20 minutes each, two cylinders of paraffin wax for 30 minutes each, embedded, sectioned at 4 microns, baked, and stained.
  • the results are shown in Table 4-Table 5.
  • the atrophy percentage and calcification area of the healthy side relative to the injured side in the treatment group were significantly lower than those in the model group at 8 weeks after operation (P ⁇ 0.01).
  • a total of 80 SPF male SD rats with a body weight of 220-240 g were selected.
  • the experimental animals had free access to food and water.
  • the experimental animals were anesthetized and maintained with isoflurane gas. Fix the supine position, incise the skin along the midline of the neck, expose the right common carotid artery, carefully separate the nerves and fascia from the bifurcation of the common carotid artery to the vessels around the base of the skull, and separate the branches of the external carotid artery from the occipital artery, superior thyroid artery, tongue Artery and maxillary maxillary artery, ligated and severed.
  • the experimental group 1 5 days after the administration it showed that it had a comparable effect on repairing the nerve damage caused by stroke (P ⁇ 0.05) compared with the positive control 1 and the positive control 2; Nerve damage repair effect (P ⁇ 0.05); Compared with before administration, the experimental group 2 had a more significant effect of repairing the nerve damage after stroke than the positive control 1 and positive control 2 at 5 days after administration (P ⁇ 0.01 ).

Abstract

本发明涉及一种神经修复蛋白组合物，其制备包括如下步骤：在细胞蛋白提取物中加入20U/mL-35U/mL的核酸酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解15min-40min，将制得的酶解液分离纯化，即得。本发明的神经修复蛋白组合物具有细胞修复及修复神经损伤等功效，可用于修复中枢神经损伤、神经退行性疾病、脑卒中、脑外伤、共济失调、脑溢血、阿尔兹海默症、帕金森症、老年痴呆等病症或其并发症所致的神经损伤，并具有稳定性好、生物利用度高、安全有效、便于生产和储运等优点。

Description

一种神经修复蛋白组合物及其制备方法和其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种神经修复蛋白组合物及其制备方法和其应用。
背景技术
细胞是生命活动的基本单位和机体健康的基础。当机体的氧化还原平衡遭到破坏时，会引起氧化还原信号和控制的中断，引发氧化应激损伤而引发多种疾病。脑部缺血缺氧(如脑卒中、脑溢血、长期高原作业、颈内动脉及椎动脉闭塞和/或狭窄等)及氧化应激损伤等，可引发多种因神经元或胶质细胞的丢失而导致的神经退行性疾病，包括记忆力减退、阿尔兹海默症、帕金森症、老年痴呆、多发性硬化症、共济失调、亨廷顿病、肌萎缩侧索硬化症等。目前，我国帕金森患者超过300万，渐冻症患者超过20万，老年痴呆患者超过千万，且随着全球人口老年化的加剧而存在高发病率等特点。及时修复损伤细胞可以显著改善、治疗相关病变。
脑卒中(脑供血动脉狭窄或闭塞导致血液不能流入大脑)及脑溢血(脑部血管突然破裂)为常见的中枢神经系统疾病，因脑供血不足所致脑组织缺血缺氧性损伤，并表现出相应神经功能缺损症状，包括平衡问题、偏瘫、感觉和振动感觉丧失、麻木、反射减弱、上睑下垂、视野缺陷、失语和失用等，过量的自由基是早期急性缺血性卒中缺血 再灌注损伤加重的主要原因，该病变具有发病率高、疾病预后差、易复发、致残率高、死亡率高等特点，严重影响人类的健康和生存质量。共济失调多由小脑、本体感觉及前庭功能障碍导致的运动笨拙和不协调，累及躯干、四肢和咽喉肌时可引起身体平衡、姿势、步态及言语障碍，包括小脑性共济失调、大脑性共济失调、感觉性共济失调、前庭性共济失调等，病因复杂且常为多种疾病的综合表现，包括由梗死、水肿或出血导致的急性脑肿胀等。脑外伤多由外物造成的头脑部外伤伤害，常引起不同程度的永久性功能障碍，包括运动、感觉、言语、视觉、听觉等区域异常局灶性症状。周围神经损伤主要由创伤、肿瘤及代谢性疾病(如糖尿病及其并发症)等因素引起的临床常见病，经常会导致机体受累节段运动、感觉和自主功能的部分或全部丧失，甚至出现顽固性神经痛，严重影响患者的生活质量。WHO于2017年发布《全球糖尿病报告》显示，全球糖尿病患病人数呈现逐年增加趋势。我国糖尿病患者1.29亿，其多种并发症(包括周围神经及末梢神经缺血缺氧引起的神经痛、神经损伤等)导致糖尿病患者致残致死等严重后果。
脑卒中的临床治疗在溶栓的同时强调脑神经保护，以期逆转神经元凋亡并改善神经损伤。丁苯酞可降低细胞内钙离子浓度，抑制谷氨酸释放并减少花生四烯酸生成、清除氧自由基、提高氧化酶活性等，作用于脑缺血病变的多个病理环节，临床将其用于改善脑梗死病灶局部循环，减小梗死面积，减轻脑组织损伤，恢复神经功能，用于急性脑缺血性脑卒中患者神经功能缺损的改善，主要用于治疗轻、中度急 性缺血性脑卒中。依达拉奉右莰醇为抗氧化剂和自由基清除剂，可清除多种自由基，减轻脑水肿和组织损伤，抑制脑缺血再灌注导致的炎性因子表达而减少细胞凋亡和细胞坏死，临床将其用于改善急性脑梗塞所致的神经症状、日常生活活动能力和功能障碍等。
细胞及微生物受到外界刺激及外源应激物(包括冷、热、酸、碱、电流、辐射、化学物质等)应激诱导时会因应激反应而诱导细胞及微生物产生应激蛋白。文献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)公开了从芸香科植物(Atalantiamonophylla)提取制得的化合物1-16具有抑制肿瘤细胞生长等活性。
间充质干细胞(mesenchymal stemcells，MSCs)具有自我复制和多向分化潜能，广泛存在于骨髓、脂肪、滑膜、牙髓、羊水、胎盘、脐带、胚胎、脐带血、羊膜、外周血、肌肉、尿液等组织，具有来源广泛、无需配型、感染率低、分化潜能强、增殖能力强、采集方便等特点，可产生干细胞生长因子(SCF)、神经生长因子(NGF)、白介素-6(IL-6)、白介素-7(IL-7)、肿瘤坏死因子(TNF)、干扰素(IFN)等活性因子，参与调节细胞生长、细胞凋亡、细胞分化、抗病毒、免疫成熟等过程，可用于免疫调节、组织修复及治疗急性肺损伤、重症肺炎、急性呼吸窘迫综合征等疾病。但MSCs产品需在其生产、储运和应用等环节采用冷藏方式，且其细胞活力保持≤12h，而限制其治疗应用。为此，需要开发安全有效的神经修复药物以满足临床需求。
发明内容
本发明的目的在于提供一种神经修复蛋白组合物，其制备包括下述步骤：
(1)在本发明的细胞蛋白提取物中加入20U/mL-35U/mL的核酸酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解15min-40min，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成5-15mg/ml后，过色谱柱，洗脱流速为0.1-1ml/min，监测并收集紫外波长为280nm的洗脱馏分，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
本发明的优选技术方案中，所述蛋白提取物的制备包括如下步骤：
S-1:将密度为5.0×106个/mL-1.0×107个/mL的间充质传代干细胞置于含有DMEM/F12 40-50％、RPMI1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养2h-6h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合，

S-2:将收集细胞按照密度为5.0×106个/mL-5.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
S-3:将步骤S-2制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
本发明的优选技术方案中，步骤S-1的培养基中含有DMEM/F12 42-45％、RPMI1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF)5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和3-8μmol/L的应激物。
本发明的优选技术方案中，步骤S-1的培养基中含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL，胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和4-6μmol/L的应激物。
本发明的优选技术方案中，步骤S-1的间充质传代干细胞密度为8.0×106-2.0×107个/mL，优选为8.0×106-1.0×107个/mL。
本发明的优选技术方案中，步骤S-1的间充质传代干细胞在培养基中培养3h-5h，优选为3.5h-4.5h。
本发明的优选技术方案中，步骤S-1中洗涤细胞的溶剂选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合,细胞洗涤次数为2-5次，优选为3-4次。
本发明的优选技术方案中，步骤S-1所述的分离选自离心、过滤的任一种或其组合，其中，所述离心条件为1000-2000rpm*3-15min，优选为1200rpm-1500rpm*5-10min。
本发明的优选技术方案中，步骤S-2的超声条件为：在2℃-8℃、25kHZ、360W条件下工作3s再间隙1s，超声处理1-5min。
本发明的优选技术方案中，步骤S-3所述的分离选自2000-8000rpm*10-30min离心、多级离心、多级过滤的任一种或其组合，优选为3000-7000rpm*15-25min。
本发明的优选技术方案中，步骤S-3的多级离心依次为3000-4000rpm*3-5min、5000-6000rpm*3-5min和7000rpm*5-8min。
本发明的优选技术方案中，所述多级过滤的滤膜孔径选自80um、50um、30um、10um、5um的任一种。
本发明的优选技术方案中，在S-3制得的细胞蛋白提取物中加入25U/mL-30U/mL的核酸酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解20min-30min，制得酶解液。
本发明的优选技术方案中，所述核酸酶选自RNA核酸酶、DNA核酸酶的任一种或其组合。
本发明的优选技术方案中，将步骤S-3制得的细胞蛋白提取物或 步骤(2)制得的蛋白组合物冻存，优选冻存于-40℃至-20℃。
本发明的优选技术方案中，在步骤S-3制得的细胞蛋白提取物或步骤(2)制得的蛋白组合物中加入冻干保护剂，冻干，制得细胞蛋白提取物冻干制剂或蛋白组合物冻干制剂，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、甘油、蔗糖、海藻糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯(HES)、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
本发明的优选技术方案中，以质量百分比计，所述冻干制剂中含有冻干保护剂0.5-8％，优选为1-5％。
本发明的优选技术方案中，在在步骤S-3制得的细胞蛋白提取物或步骤(2)制得的蛋白组合物中任选地加入蛋白稳定剂，其中，所述蛋白稳定剂选自白蛋白、锌盐、铝盐的任一种。
本发明的优选技术方案，所述细胞蛋白提取物冻干制剂或蛋白组合物冻干制剂pH6-8，优选为pH7-7.5。
本发明的优选技术方案中，所述神经修复蛋白组合物的分子量为20kDa-250kDa，优选为35kDa-200kDa。
本发明的优选技术方案，所述神经修复蛋白组合物中的蛋白组成如图2所示。
本发明的优选技术方案中，所述冻干制剂临用前用生理盐水或5％葡萄糖溶液复溶后，采用静脉注射、鞘内注射、腰椎穿刺的任一种或其组合方式使用。
本发明的优选技术方案中，所述间充质传代干细胞的培养或原代 间充质干细胞的培养采用本领域的培养方法。
本发明的优选技术方案中，所述间充质传代干细胞的培养包括下述步骤：将原代间充质干细胞按照初始密度为5.0×105-5.0×106个/ml加入到传代培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每隔2-3天，观察传代培养基变黄后，半量更换传代培养基，其中，所述传代培养基含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基。
本发明的优选技术方案中，所述原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于含10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养，每间隔2-3天半量更换培养基，培养至组织块爬出细胞；
2)振摇，收集低层细胞用PBS清洗后，加入0.25％的胰蛋白酶消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打，1200-1500rpm/min*5-8min离心后，收集细胞，即得。
本发明的目的在于提供一种具有神经修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
S-1:将密度为5.0×106个/mL-5.0×107个/mL的间充质传代干细胞置于含有DMEM/F12 40-50％、RPMI1640 40-50％、牛血清蛋白(BSA) 0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养2h-6h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合；
S-2：将收集细胞按照密度为5.0×106个/mL-5.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
S-3:将步骤S-2制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
本发明的优选技术方案中，步骤S-1的培养基中含有DMEM/F12 42-45％、RPMI1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF)5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和3-8μmol/L的应激物。
本发明的优选技术方案中，步骤S-1的培养基中含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL，胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和4-6μmol/L的应激物。
本发明的优选技术方案中，步骤S-1的间充质传代干细胞密度为8.0×106-2.0×107个/mL，优选为8.0×106-1.0×107个/mL。
本发明的优选技术方案中，步骤S-1的间充质传代干细胞在培养基中培养3h-5h，优选为3.5h-4.5h。
本发明的优选技术方案中，步骤S-1中洗涤细胞的溶剂选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合,细胞洗涤次数为2-5次，优选为3-4次。
本发明的优选技术方案中，步骤S-1所述的分离选自离心、过滤的任一种或其组合，其中，所述离心条件为1000-2000rpm*3-15min，优选为1200rpm-1500rpm*5-10min。
本发明的优选技术方案中，步骤S-2的超声条件为：在2℃-8℃、25kHZ、360W条件下工作3s再间隙1s，超声处理1-5min。
本发明的优选技术方案中，步骤S-3所述分离选自2000-8000rpm*10-30min离心、多级离心、多级过滤的任一种或其组合，优选为3000-7000rpm*15-25min。
本发明的优选技术方案中，步骤S-3的多级离心依次为3000-4000rpm*3-5min、5000-6000rpm*3-5min和7000rpm*5-8min。
本发明的优选技术方案中，所述多级过滤的滤膜孔径选自80um、50um、30um、10um、5um的任一种。
本发明的优选技术方案中，将步骤S-3制得的细胞蛋白提取物冻存，优选冻存于-40℃至-20℃。
本发明的优选技术方案中，将步骤S-3制得的细胞蛋白提取物采用核酸酶或全能核酸酶的任一种酶解后再分离纯化。
本发明的优选技术方案中，所述间充质传代干细胞的培养或原代间充质干细胞的培养采用本领域的培养方法。
本发明的优选技术方案中，所述间充质传代干细胞的培养包括下述步骤：将原代间充质干细胞按照初始密度为5.0×105-5.0×106个/ml加入到传代培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每隔2-3天，观察传代培养基变黄后，半量更换传代培养基，其中，所述传代培养基含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基。
本发明的优选技术方案中，所述原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于含10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养，每间隔2-3天半量更换培养基，培养至组织块爬出细胞；
2)振摇，收集低层细胞用PBS清洗后，加入0.25％的胰蛋白酶消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打，1200-1500rpm/min*5-8min离心后，收集细胞，即得。
本发明的目的在于提供一种神经修复蛋白组合物的制备方法，包括如下步骤：
(1)在本发明的细胞蛋白提取物中加入20U/mL-35U/mL的核酸 酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解15min-40min，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成5-15mg/ml后，过色谱柱，洗脱流速为0.1-1ml/min，监测并收集紫外波长为280nm的洗脱馏分，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
本发明的优选技术方案中，所述核酸酶选自RNA核酸酶、DNA核酸酶的任一种或其组合。
本发明的优选技术方案中，在本发明的细胞蛋白提取物中加入25U/mL-30U/mL的核酸酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解20min-30min，制得酶解液。
本发明的优选技术方案中，所述神经修复蛋白组合物的分子量为20kDa-250kDa，优选为35kDa-200kDa。
本发明的优选技术方案中，将步骤(2)所得蛋白组合物冻存，优选冻存于-40℃至-20℃。
本发明的优选技术方案中，在步骤(2)收集的蛋白组合物中加入冻干保护剂，冻干，制得蛋白组合物冻干制剂，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、甘油、蔗糖、海藻糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯(HES)、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
本发明的优选技术方案中，以质量百分比计，所述冻干制剂中含 有冻干保护剂0.5-8％，优选为1-5％。
本发明的优选技术方案中，将步骤(2)收集的蛋白组合物中任选地加入蛋白稳定剂，其中，所述蛋白稳定剂选自白蛋白、锌盐、铝盐的任一种。
本发明的优选技术方案，所述冻干制剂pH6-8，优选为pH7-7.5。
本发明的优选技术方案，所述神经修复蛋白组合物中的蛋白组成如图2所示。
本发明的优选技术方案中，所述冻干制剂临用前用生理盐水或5％葡萄糖溶液复溶后，采用静脉注射、鞘内注射、腰椎穿刺的任一种或其组合方式使用。
本发明的另一目的在于提供一种神经修复组合物，所述组合物由本发明的具有神经功效的细胞蛋白提取物或神经修复蛋白组合物的任一种或其组合和药学上可接受的载体组成。
本发明的药学上可接受载体的用量或其种类根据组合物中有效成分的理化性质和含量、制剂类型、制剂的溶出及生物利用度等因素而定。
本发明的组合物可为本领域的剂型，并可采用本领域的制剂技术制备得到。
本发明的优选技术方案中，所述组合物选自冻干剂、凝胶剂、鼻喷剂、液体敷料、注射剂、贴剂、膏剂、霜剂、乳剂、栓剂的任一种。
本发明的优选技术方案中，所述组合物的给药方式选自静脉注射、 鞘内注射、腰椎穿刺的任一种或其组合。
本发明的另一目的在于提供本发明具有神经细胞修复功效的细胞蛋白提取物、神经修复蛋白组合物或其组合物用于制备细胞修复、神经修复的任一种的制品中的应用。
本发明的优选技术方案中，所述神经修复选自中枢神经损伤、神经退行性疾病、脑卒中、脑外伤、共济失调、脑溢血、阿尔兹海默症、帕金森症、老年痴呆、多发性硬化症、亨廷顿病、肌萎缩侧索硬化症、周围性神经损伤、糖尿病、记忆力减退中的任一种病症或其并发症所致的神经损伤。
本发明的优选技术方案中，所述共济失调选自小脑性共济失调、大脑性共济失调、感觉性共济失调、前庭性共济失调的任一种或其并发症。
本发明的另一目的在于提供化合物1-16用于应激诱导干细胞产生具有修复功效的功能蛋白的应用。
本发明的优选技术方案中，所述修复选自细胞修复、毛囊修复、关节修复、神经修复中的任一种。
除非另有说明，本发明涉及液体与液体之间的百分比时，所述的百分比为体积/体积百分比；本发明涉及液体与固体之间的百分比时，所述百分比为体积/重量百分比；本发明涉及固体与液体之间的百分比时，所述百分比为重量/体积百分比；其余为重量/重量百分比。
除非另有说明，本发明间充质干细胞MSCs鉴定参照《Standards  for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application》。
本发明采用商购试剂盒检测活性氧(IOD)、超氧化物歧化酶SOD、丙二醛MDA水平。
与现有技术相比，本发明具有下述有益效果：
1、本发明科学筛选含有应激物的培养基应激诱导间充质干细胞产生具有细胞修复及神经修复功效的功能蛋白，所得的细胞蛋白提取物、蛋白组合物或其组合物具有细胞修复及修复神经损伤等功效，可用于修复中枢神经损伤、神经退行性疾病、脑卒中、脑外伤、共济失调、脑溢血、阿尔兹海默症、帕金森症、老年痴呆、多发性硬化症、亨廷顿病、肌萎缩侧索硬化症、周围性神经损伤、糖尿病、记忆力减退、小脑性共济失调、大脑性共济失调、感觉性共济失调、前庭性共济失调中的任一种病症或其并发症所致的神经损伤，并具有纯度高、稳定性好、生物利用度高、安全有效、便于生产和储运等优点。
2、本发明的制备方法具有操作简便，绿色环保，成本更优，适用于工业化生产等优点。
附图说明
图1本发明的神经修复蛋白组合物的电泳分离结果；
图2本发明的神经修复蛋白组合物的高效液相检测结果；
图3本发明的细胞蛋白提取物对UVB照射所致皮肤损伤的修复作 用研究；
图4本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中活性氧化物(IOD)生成的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图5本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中超氧化物歧化酶SOD生成的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图6本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中MDA水平的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图7本发明神经修复蛋白组合物对缺血性脑卒中的损伤修复作用研究中治疗组与模型组的每周体重比较结果；
图8本发明神经修复蛋白组合物对缺血性脑卒中的损伤修复作用研究中治疗组与模型组的TTC染色结果；
图9本发明神经修复蛋白组合物对缺血性脑卒中的损伤修复作用研究中治疗组与模型组的iba1阳性细胞免疫荧光染色结果；
图10本发明神经修复蛋白组合物对缺血性脑卒中的损伤修复作用研究中治疗组与模型组的β3Tubulin阳性细胞免疫荧光染色结果；
图11本发明神经修复蛋白组合物对缺血性脑卒中损伤修复作用研究 中的神经功能(NDS)评分结果。
具体实施方式
下面结合具体实施例对本发明的详细内容做进一步解释和描述，但并不以此限制本发明的保护范围。
1、原代间充质干细胞的培养
原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于培养瓶中，加入含10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基，再将其置于37℃、5％CO2条件下培养，促进其贴壁，每间隔2-3天,观察培养基变黄后，半量更换培养基，培养10-12天，至组织块边上可见细胞爬出；
2)轻轻摇晃，使组织块掉落，分别收集组织块和低层细胞，其中，将收集的组织块再贴壁培养；
3)将收集的低层细胞用PBS清洗后，加入适量0.25％胰蛋白酶消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打瓶底，1500rpm/min*5min离心后，收集细胞，即得。
2、原代间充质干细胞的传代培养(间充质传代干细胞的培养)
原代间充质干细胞的传代培养(间充质传代干细胞的培养)：将原代间充质干细胞按照初始密度为5.0×105-5.0×106个/ml加入到含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基 中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每间隔2-3天，观察培养基变黄后，半量更换培养基。
3、化合物1-16的制备参照文献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)。
实施例1本发明具有神经修复功效的细胞蛋白提取物的制备
本发明具有神经修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为8.0×106个/mL加入到含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物16的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为1.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。可根据需要，将制得的细胞蛋白提取物冻存于-40℃至-20℃。
实施例2本发明神经修复蛋白组合物的制备
本发明神经修复蛋白组合物的制备包括如下步骤：
(1)在实施例1制得的细胞蛋白提取物中，加入25U/mL的全能核酸酶(UCF.ME UltraNuclease)，将其置于37℃酶解30min后，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成10mg/ml，依次过高纯硅胶液相色谱保护柱(WondaGuard C18，4.6×5mm)、高纯硅胶液相色谱制备柱(SHIMSENAnkylo C18，5μm，4.6×250mm)，洗脱流速为0.1-1ml/min，监测并收集紫外波长为280nm的洗脱馏分，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
实施例3本发明神经修复蛋白组合物冻干制剂的制备
在实施例2制得的细胞蛋白组合物中加入甘露醇，搅拌，混合均匀后，冻干，所得冻干制剂中含有2.15％的甘露醇(m/m)。
实施例4本发明具有神经修复功效的细胞蛋白提取物的制备
本发明具有神经修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为1.0×107个/mL加入到含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞 生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和8μmol/L的化合物13的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为2.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例5本发明神经修复蛋白组合物的制备
本发明神经修复蛋白组合物的制备包括如下步骤：
(1)在实施例4制得的细胞蛋白提取物中，加入30U/mL的全能核酸酶(UCF.ME UltraNuclease)，将其置于37℃酶解30min后，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成10mg/ml，依次过高纯硅胶液相色谱保护柱(WondaGuard C18，4.6×5mm)、高纯硅胶液相色谱制备柱(SHIMSENAnkylo C18，5μm，4.6×250mm)，洗脱流速为0.5ml/min，监测并收集紫外波长为280nm的蛋白组合物，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
实施例6本发明神经修复蛋白组合物冻干制剂的制备
在实施例5制得的细胞蛋白组合物中加入右旋糖酐，搅拌，混合均匀后，冻干，所得冻干制剂中含有1％的右旋糖酐(m/m)。
实施例7本发明具有神经修复功效的细胞蛋白提取物的制备
本发明具有神经修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为9.0×106个/mL加入到含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物14的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为2.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例8本发明神经修复蛋白组合物的制备
本发明神经修复蛋白组合物的制备包括如下步骤：
(1)在实施例7制得的细胞蛋白提取物中，加入20U/mL的全能 核酸酶(UCF.ME UltraNuclease)，将其置于37℃酶解30min后，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成10mg/ml，依次过高纯硅胶液相色谱保护柱(WondaGuard C18，4.6×5mm)、高纯硅胶液相色谱制备柱(SHIMSENAnkylo C18，5μm，4.6×250mm)，洗脱流速为0.1-1ml/min，监测并收集紫外波长为280nm的蛋白组合物，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
实施例9本发明神经修复蛋白组合物冻干制剂的制备
在实施例8制得的细胞蛋白组合物中加入山梨醇，搅拌，混合均匀后，冻干，即得，所得冻干制剂中含有5％的山梨醇(m/m)。
实施例10本发明具有神经修复功效的细胞蛋白提取物的制备
本发明具有神经修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为1.0×107个/mL加入到含有DMEM/F12 45％、RPMI1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和6μmol/L的化合物15的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为2.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例11本发明神经修复蛋白组合物的制备
本发明神经修复蛋白组合物的制备包括如下步骤：
(1)在实施例10制得的细胞蛋白提取物中，加入30U/mL的全能核酸酶(UCF.ME UltraNuclease)，将其置于37℃酶解30min后，制得酶解液；
(2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成10mg/ml，依次过高纯硅胶液相色谱保护柱(WondaGuard C18，4.6×5mm)、高纯硅胶液相色谱制备柱(SHIMSENAnkylo C18，5μm，4.6×250mm)，洗脱流速为1ml/min，监测并收集紫外波长为280nm的蛋白组合物，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
实施例12本发明神经修复蛋白组合物冻干制剂的制备
在实施例11制得的细胞蛋白组合物中加入甘露醇，搅拌，混合均匀后，冻干，即得，所得冻干制剂中含有3％的甘露醇(m/m)。
实施例13本发明细胞蛋白提取物冻干制剂的制备
在实施例1制得的细胞蛋白提取物加入所需量的甘露醇，搅拌，混合均匀后，冻干，所得冻干制剂中含有5％的甘露醇(m/m)。
实施例14本发明神经修复蛋白组合物的分子量分布检测
采用标准分子量聚丙烯酰胺凝胶电泳检测本发明神经修复蛋白组合物分子量分布，包括下述步骤：
1、选取厚度为1.5mm的玻璃板水平放置，将按照表1配置好的胶液(15％的下层胶、4％的分离胶)依次平铺至玻璃板上，将梳子垂直插入至下层胶上；
表1
试剂 4％的分离胶 15％的下层胶
ddH2O 6ml 3.7ml
30％丙烯酰胺混合液 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
2、将冻存于-40℃的Marker(20KDa-245KDa)解冻后，将Marker和实施例3、实施例6、实施例9、实施例12的神经修复蛋白组合物冻干粉分别用PBS配置成10ug/ul的待测样品，添加20ul待测样品至上样孔后，在60-80V条件下电泳至出现较为清晰的条带后，调整电压为100-120V，至Marker分离完全，将PAGE胶置于考马斯亮蓝染液中，待凝胶上出现清晰的条带时，停止染色。用纯水清洗后，用 10％的醋酸溶液脱色，待凝胶变透明时停止脱色。结果见图1，其中，条带A为实施例3、条带B为实施例6、条带C为实施例9、条带D为实施例12。
实施例15本发明神经修复蛋白组合物的高效液相色谱检测
将实施例3的神经修复蛋白组合物冻干粉用去离子水溶解后，将其制成10mg/ml的供试品溶液。
色谱柱：SHIMSEN Ankylo(300mm*4.6mm.D.,3um；P/N:380-01215-05)岛津
流动相：50mmol/L磷酸盐缓冲液(pH＝6.8)，含300mmol/L氯化钠；流速：0.3mL/min；进样量：10ul；柱温：25℃；检测波长：280nm；等度洗脱，采集30min。结果见图2。
试验例1本发明的细胞蛋白提取物对UVB辐射损伤皮肤的修复作用研究
3D皮肤模型：商购于EpiKutis。
模型培养液：DMEM基础培养基。
VE工作液：吸取0.5gVE原液，将其溶于10mL无水乙醇中，配制成5％的母液；吸取100μL5％母液，将其溶于10mL模型培养液中，配制成0.05％的VE工作液。
在6孔板中加入0.9mL模型培养液，将3D皮肤模型转移到6孔板中，标注测试编号。
空白对照(BC)：皮肤模型不做任何处理，将其置于CO2培养箱(37℃、5％CO2)中孵育48h；
阴性对照(NC)：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；加入25μL 0.2％的SLS工作液，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
阳性对照(PC)：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；添加25ul的0.05％的VE工作液，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
试验组：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；添加1％的细胞蛋白提取物溶液25ul(将实施例13制得的细胞蛋白提取物冻干粉用生理盐水制成1％的溶液)，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h。
孵育48h后，用PBS溶液洗涤并清除皮肤模型内外残留液体。用4％的多聚甲醛固定处理24h后，将模型环切取下，经H&E染色处理后观察，结果见图3。检测UVB辐射损伤皮肤的活性氧(ROS)、超氧化物歧化酶、丙二醛(MDA)水平，结果见图4-图6。本发明的细胞蛋白提取物对UVB辐射损伤皮肤具有明显的修复作用。
试验例2本发明神经修复蛋白组合物对缺血性脑卒中损伤的修复作用考察
选取6-7周的SPF级雄性SD大鼠75只。试验动物用普通饲料适应性喂养1周后，所有动物进行脑缺血再灌注手术，构建脑卒中模型。 试验动物再灌注结束后，待其麻醉苏醒，即刻进行神经功能评分。
试验大鼠在实验前一天禁食一晚，麻醉后呈仰卧位固定于手术板上，颈部纵行切口约3cm，暴露并钝性游离颈总动脉(CCA)、颈外动脉(ECA)、颈内动脉(ICA)，于颈总动脉(CCA)近心端打活结，暂时阻断血供，颈外动脉(ECA)打双结，并在双结中间用显微剪剪开小口穿入线栓，从双结中间离断颈外动脉(ECA)，调整线栓方向使之进入颈内动脉(ICA)，以颈内动脉(ICA)和颈外动脉(ECA)分叉处为参考位置，线栓插入到预设刻度，感到阻力，提示线栓头部已到达大脑中动脉(MCA)，打结固定线栓。缺血60min，将线栓拔出，松掉颈总动脉活结，完成。
试验动物术后存活62只。根据评分结果，将试验动物分为模型组(30只)和治疗组(32只)，且均进行侧脑室注射3次，包括脑缺血再灌注后即刻给药、术后1周给药、术后2周给药。治疗组(实施例3的神经修复蛋白组合物冻干粉用生理盐水溶解,给药剂量为6.77mgf/kg，给药体积20ul)，模型组对应侧脑室给与同体积生理盐水。术后3天，各组选取2只动物进行TTC染色；术后4周，模型组取材15只，治疗组取材16只，各组选取1只试验动物进行TTC染色；术后8周，模型组取材13只，治疗组取材14只，各组选取1只试验动物进行TTC染色。
试验期间,每周观察试验动物1次，包括死亡情况、精神状态、行为活动、发病、呼吸、分泌物、粪便、饮食和饮水情况。每周检测所有试验动物体重1次，结果见图7。试验期间，治疗组与模型组的 体重未显现差异性。
术后3天、术后4周、术后8周进行脑卒中模型一致性评价。取血后，立即从左心室插管，快速灌注生理盐水冲洗2min，断头取脑，作TTC染色，将大脑从前往后切成5-7个冠状切片，片厚度约为1.8mm，用1％TTC于37℃染色10min后，用组织固定液固定后，应用扫描仪进行扫描，并用分析软件进行分析。与正常组织中的脱氢酶反应而呈红色，而缺血组织内脱氢酶活性下降而不能反应，故不会产生变化呈苍白，结果见图8。
采用动物四肢抓力检测仪检测评价术前、术后1周的动物肌肉力量。采用转棒疲劳仪检测试验动物术前、术后1周的全身耐力：转速12r/min，连续进行3次实验，每次3min，包括跌落时速度、跑步持续实验、行进路程的3次均值，结果见表2。治疗组术后1周前肢抓力、跌落前速度、行进路程、持续时间均高于模型组(P<0.01)。
表2


注：与模型组相比，*表示P<0.05,**表示p<0.01。
水迷宫测试试验动物术后4周、术后8周的动物学习记忆功能及活跃度评价：前四天先进行获得性训练实验，第五天进行平台探索实验。获得性训练实验方法：先将平台露出水面并使得大鼠可以看见平台。再将大鼠放入泳池，如大鼠毫无困难地直接游向平台，说明其游泳能力和视力均正常，可以开始实验。将平台安装在第二象限水面下5mm处，大鼠背向池壁并放入水中，其放入位置随机不重复取1、2、3、4四个象限起始位置之一，4个象限各放入1次，共计4次，记录大鼠找到水下平台的时间(逃避潜伏期)。如果逃避潜伏期超过60s，则引导大鼠到平台，让其在平台上停留10s后将大鼠移开并擦干。必要时，将大鼠放在150W的白炽灯下烤5min，放回笼内。平台探索实验方法：在获得性训练结束后，第二天将第二象限的平台取掉，在四号象限起始位置，大鼠头朝池壁放入水中。实验结束后，对获得性实验的站台穿越次数进行分析，结果见表3。治疗组术后4周、术后8周穿越平台次数均高于模型组(P<0.01)。
表3

注：与模型组相比，*表示P<0.05,**表示p<0.01。
病理检测：组织样本取材3mm厚，梯度酒精脱水70％、80％、95％、 100％各30min，二甲苯两瓶各20min，石蜡浸蜡两缸各30min，包埋，切片4微米，烤片，染色。结果见表4-表5。治疗组术后8周健全侧相对于损伤侧的萎缩百分比和钙化面积均明显低于模型组(P<0.01)。
表4

注：与模型组相比，*表示P<0.05,**表示p<0.01。
表5

注：与模型组相比，*表示P<0.05,**表示p<0.01。
免疫荧光染色结果(见表6、图9-图10)。治疗组术后4周、术后8周的iba1阳性细胞面积明显低于模型组(p<0.05)；治疗组术后8周的β3Tubulin阳性细胞个数明显高于模型组(p<0.01)。
表6

注：与模型组相比，*表示P<0.05,**表示p<0.01。
试验例3本发明神经修复蛋白组合物对缺血性脑卒中的损伤修复作用研究
选取体重为220-240g的SPF级SD雄性大鼠80只。试验动物自由采食和饮水。
试验动物用异氟烷气体麻醉并维持。仰卧位固定，沿颈正中线切开皮肤，暴露右侧颈总动脉，小心分离颈总动脉分叉至颅底部血管周围的神经及筋膜，分离颈外动脉分支枕动脉、甲状腺上动脉、舌动脉和上颚上颌动脉，结扎并切断。从颈外动脉远心端将3#尼龙线(直径0.25mm)导入到颈内动脉，插至Willis环大脑中动脉处，以有效阻断大脑中动脉，插入的尼龙线长度距颈总动脉分叉处18～20mm。MCAO后2h，小心抽出颈内动脉血管腔内的尼龙线使颈内动脉再灌注，动物清醒完全后进行神经功能评分(NDS，见表7)。试验动物评分>8分为造型成功。
表7神经功能评分标准

选取造模成功的60只大鼠按照评分数随机分为6组，每组10只，给药方式见表8)。试验动物再灌注后静脉滴注给药。
表8
试验动物在给药前、给药后1天、3天、5天采用盲法对动物进行神经功能评分。试验数据以均值±标准差(Mean±SEM)表示，用SPSS 25.0软件进行统计分析。方差齐做单因素方差分析，有显著差异时采用LSD检验进行两两比较；方差不齐做Kruskal-Wallis非参数检验，并进行两两比较。结果见图11。与给药前相比，试验组1 在给药后5d，显示具有与阳性对照1、阳性对照2相当的修复脑卒中神经损伤效果(P<0.05)；与给药前相比，试验组2在给药后3d，显示具有显著的神经损伤修复效果(P<0.05)；与给药前相比，试验组2在给药后5d，具有较与阳性对照1、阳性对照2更为显著的修复脑卒中神经损伤效果(P<0.01)。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明权利要求保护的范围。

Claims (10)

  1. 一种神经修复蛋白组合物，其制备包括下述步骤：
    (1)在本发明的细胞蛋白提取物中加入20U/mL-35U/mL的核酸酶或全能核酸酶的任一种或其组合，将其置于37℃±1℃条件下酶解15min-40min，制得酶解液；
    (2)在2℃-8℃条件下，将步骤(1)制得的酶解液用洗脱溶剂配置成5-15mg/ml后，过色谱柱，洗脱流速为0.1-1ml/min，监测并收集紫外波长为280nm的洗脱馏分，即得，其中，洗脱溶剂由50mmol/L磷酸盐缓冲液(pH6.8)中含300mmol/L氯化钠组成。
  2. 如权利要求1所述的蛋白组合物，所述蛋白提取物的制备包括如下步骤：
    S-1:将密度为5.0×106个/mL-1.0×107个/mL的间充质传代干细胞置于含有DMEM/F12 40-50％、RPMI 1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养2h-6h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合，

    S-2:将收集细胞按照密度为5.0×106个/mL-5.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
    S-3:将步骤S-2制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
  3. 如权利要求1-2任一所述的蛋白组合物，步骤S-1的培养基中含有DMEM/F12 42-45％、RPMI 1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF)5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和3-8μmol/L的应激物。
  4. 如权利要求1-3任一所述的蛋白组合物，步骤S-1的培养基中含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL，胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和4-6μmol/L的应激物。
  5. 如权利要求1-4任一所述的蛋白组合物，所述神经修复蛋白组合物的分子量为20kDa-250kDa，优选为35kDa-200kDa。
  6. 如权利要求1-5任一所述的蛋白组合物，在步骤(2)制得的蛋白组合物中加入冻干保护剂，冻干，制得蛋白组合物冻干制剂，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、海藻糖、甘油、蔗糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
  7. 如权利要求1-6任一所述的蛋白组合物，以质量百分比计，所述冻干制剂中含有冻干保护剂0.5-8％，优选为1-5％。
  8. 一种神经修复组合物，所述组合物由权利要求1-7所述的神经修复蛋白组合物和药学上可接受的载体组成。
  9. 如权利要求1-7所述的神经修复蛋白组合物或如权利要求8所述的神经修复组合物用于制备细胞修复、神经修复的任一种的制品中的应用。
  10. 化合物1-16用于应激诱导干细胞产生具有修复功效的功能蛋白的应用。
PCT/CN2023/073582 2022-01-28 2023-01-28 一种神经修复蛋白组合物及其制备方法和其应用 WO2023143522A1 (zh)

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