Nerve repair protein compositions and methods for making and using same

Abstract

The present invention relates to a nerve repair protein composition, the preparation of which includes the steps of adding 20 U / mL to 35 U / mL of any one or a combination of nuclease or ultranuclease to a cell protein extract, performing enzymatic degradation for 15 to 40 minutes at 37°C ± 1°C, and isolating and purifying the produced enzymatic degradation solution. The nerve repair protein composition of the present invention has effects such as cell repair and nerve damage repair, and can be used to repair nerve damage caused by symptoms such as central nerve damage, neurodegenerative disease, stroke, brain trauma, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, dementia, or complications thereof, and has the advantages of good stability, high bioavailability, safety, effectiveness, and easy production, storage, and transportation.

Description

[Technical field]

[0001] The present invention relates to the field of biomedical technology, specifically to neural repair protein compositions and methods for making and using same. [Background technology]

[0002] Cells are the basic unit of life activity and the basis of the health of living organisms. When the redox balance of living organisms is destroyed, it causes the interruption of redox signals and control, inducing oxidative stress disorders and inducing various diseases. Ischemic hypoxic encephalopathy (e.g., stroke, intracerebral hemorrhage, long-term work at high altitude, occlusion and / or stenosis of the internal carotid artery and vertebral artery, etc.) and oxidative stress disorders can induce various neurodegenerative diseases caused by the loss of neurons or glial cells, including memory loss, Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, ataxia, Huntington's disease, amyotrophic lateral sclerosis, etc. At present, there are 3 million Parkinson's disease patients, 200,000 amyotrophic lateral sclerosis patients, and more than 10 million dementia patients in China, and with the aging of the world's population, there are characteristics such as a high incidence rate. Timely repair of damaged cells can significantly improve and treat related pathologies.

[0003] Stroke (narrowing or occlusion of the arteries supplying the brain with blood, preventing blood from entering the brain) and intracerebral hemorrhage (sudden rupture of a blood vessel in the brain) are common central nervous system disorders in which insufficient blood supply to the brain leads to hypoxic-ischemic damage to brain tissue, with corresponding symptoms of neurological dysfunction including balance problems, hemiplegia, loss of sensory and vibration sensation, numbness, reduced reflexes, ptosis, visual field defects, aphasia and apraxia, etc. Excessive free radicals are a factor in aggravating ischemia-reperfusion injury in the early stage of acute ischemic stroke, and such lesions have characteristics such as high incidence rate, poor prognosis of the disease, easy recurrence, high disability rate, and high mortality rate, which have a significant impact on human health and quality of life. Ataxia is often caused by cerebellar, proprioceptive and vestibular dysfunction, resulting in unnatural and uncomfortable movements. When the trunk, limbs and pharyngeal muscles are affected, it can cause body balance, posture, gait and speech disorders. It includes cerebellar ataxia, cerebral ataxia, sensory ataxia, vestibular ataxia, etc., and has a complex etiology and is often a comprehensive expression of various diseases, including acute brain swelling due to infarction, edema or hemorrhage. Brain damage is often caused by head traumatic injury by external objects, often causing permanent functional disorders of different degrees, and includes abnormal focal symptoms in areas such as motor, sensory, speech, vision and hearing. Peripheral nerve damage is a common clinical disease mainly caused by factors such as trauma, tumors and metabolic diseases (e.g. diabetes and its complications), and often affects the organism, resulting in partial or total loss of segmental motor, sensory and autonomic functions, and even leading to intractable neuralgia, which seriously affects the quality of life of patients. The World Diabetes Report published by the WHO in 2017 showed that the number of diabetes patients worldwide is increasing year by year. There are 129 million diabetes patients in China, and various complications (including neuralgia and nerve damage caused by peripheral nerve and peripheral ischemic hypoxia) can lead to serious consequences such as physical disability and death for diabetic patients.

[0004] In the clinical treatment of stroke, in order to reverse the consequences of neuronal apoptosis and improve neuronal damage, together with the dissolution of thrombus, the protection of the brain nerves is also emphasized. DL-3-n-butylphthalide can reduce the concentration of intracellular calcium ions, inhibit the release of glutamate, reduce the production of arachidonic acid, scavenge oxygen radicals, improve oxidase activity, etc., and act on multiple pathological stages of cerebral ischemic lesions. In clinical practice, it is used to improve the local circulation of cerebral infarction lesions, reduce the infarct area, reduce brain tissue damage, and restore neurological function, and is used to improve the neurological dysfunction of patients with acute cerebral ischemic stroke, mainly used in the treatment of mild and moderate acute ischemic stroke. Edaravone (Edaravone Dexborneol) is an antioxidant and free radical scavenger that scavenges various free radicals, reduces cerebral edema and tissue damage, inhibits the expression of inflammatory factors caused by cerebral ischemia-reperfusion, and reduces apoptosis and cell necrosis. In clinical practice, it is used to improve neurological symptoms, daily living activity ability, and functional disorders caused by acute cerebral infarction.

[0005] When cells and microorganisms are subjected to stress induction by external stimuli and exogenous stress substances (including cold, heat, acid, alkali, electric current, radiation, chemical substances, etc.), the cells and microorganisms are induced to produce stress proteins through a stress response. Literature 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41: 431-437) discloses that compounds 1 to 16 prepared by extraction from a citrus plant (Atalantia monophylla) have activity to inhibit the proliferation of tumor cells, etc.

[0006] Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate in multiple directions. They are widely present 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. They have characteristics such as wide source, no need for matching, low infection rate, strong differentiation ability, strong proliferation ability, and easy collection. They can produce activating factors such as stem cell factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., and are involved in the regulation of processes such as cell proliferation, apoptosis, cell differentiation, antiviral, and immune maturation. 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 during production, storage, transportation, and use, and their cell activity can only be maintained for 12 hours or less, limiting their therapeutic use. Therefore, there is a need to develop a safe and effective neuroderma repair drug to meet clinical needs. Summary of the Invention [Problem to be solved by the invention]

[0007] The object of the present invention is to provide a neural repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting mixture to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) adjusting the enzymatic degradation solution produced in step (1) to 5 to 15 mg / ml at 2°C to 8°C with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride, and then passing the solution through a chromatography column to monitor and collect the eluted fraction having an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0008] In a preferred technical solution of the present invention, the preparation of the protein extract comprises: Density is 5.0×10 6 pieces / mL~1.0×10 7 Step S-1: placing mesenchymal passaged stem cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium for 2h-6h under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, followed by separating, washing, and collecting the cells; [Table 1-1] [Table 1-2] The collected cells were diluted to 5.0 × 10 6 pieces / mL~5.0×10 7 Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0009] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0010] In a preferred technical proposal of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 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% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0011] In a 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 5.0×10 6 ~1.0×10 7 pieces / mL.

[0012] In a preferred technical solution of the present invention, the mesenchymal passaged stem cells in step S-1 are cultured in the medium for 3 to 5 hours, preferably 3.5 to 4.5 hours.

[0013] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of cell washing is 2 to 5 times, preferably 3 to 4 times.

[0014] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0015] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0016] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0017] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0018] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0019] In a preferred technical solution of the present invention, 25 U / mL to 30 U / mL of either nuclease or ultranuclease or a combination thereof is added to the cell protein extract prepared in S-3, and the mixture is subjected to enzymatic hydrolysis at 37°C±1°C for 20 to 30 minutes to prepare an enzymatic hydrolysis solution.

[0020] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0021] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0022] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 or the protein composition prepared in step (2) is added with a freeze-drying protection agent selected from any one or combination of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch, and then freeze-dried to prepare a cell protein extract freeze-dried preparation or a protein composition freeze-dried preparation.

[0023] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass.

[0024] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the cellular protein extract prepared in step S-3 or the protein composition prepared in step (2), which is any one selected from albumin, zinc salts, and aluminum salts.

[0025] In a preferred technical solution of the present invention, the lyophilized preparation of the cell protein extract or the lyophilized preparation of the protein composition has a pH of 6 to 8, preferably 7 to 7.5.

[0026] 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.

[0027] In a preferred technical solution of the present invention, the protein composition in the nerve repair protein composition is as shown in FIG.

[0028] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted with saline or 5% glucose solution just before use, and then administered by intravenous injection, intrathecal injection, lumbar puncture or any combination thereof.

[0029] In a preferred technical solution of the present invention, the culture of the passaged mesenchymal stem cells or the culture of the primary mesenchymal stem cells is carried out by a culture method known in the art.

[0030] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The method includes the steps of adding the cells to a subculture medium, which is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, so that the initial density of the cells becomes 10 cells / ml, and then culturing the medium at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days, and replacing half of the subculture medium every 2 to 3 days after observing the yellowing of the subculture medium.

[0031] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was taken and cut into 3 mm pieces. 3 Then, the tissue pieces are cut into small pieces, centrifuged and washed to collect the tissue pieces, which are then 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℃ and 5%±1.0% CO2, changing half of the medium every 2-3 days, until cells emerge from the tissue pieces. and 2) shaking the mixture to collect the lower layer cells and washing them with PBS, then adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min for 5-8 minutes, and collecting the cells.

[0032] The object of the present invention is to provide a method for producing a cellular protein extract having a neurorepair effect, the method comprising: Density is 5.0×10 6 pieces / mL~5.0×10 7 Step S-1: placing mesenchymal passaged stem cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15ug / mL epidermal growth factor (EGF), 1-15ug / mL fibroblast growth factor (FGF), 1-15ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10μmol / L of any one or combination of compounds 1-16, and then culturing the medium for 2h-6h under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, followed by separating, washing, and collecting the cells; The collected cells were diluted to 5.0 × 10 6 pieces / mL~5.0×10 7 Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... and step S-3 of separating the cell lysate prepared in step S-2 and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0033] In a preferred technical proposal of the present invention, the medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L of a stress substance.

[0034] In a preferred technical proposal of the present invention, the medium in step S-1 contains 45% DMEM / F12, 45% RPMI1640, 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% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance.

[0035] In a 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 5.0×10 6 ~1.0×10 7 pieces / mL.

[0036] In a preferred technical solution of the present invention, the mesenchymal passaged stem cells in step S-1 are cultured in the medium for 3 to 5 hours, preferably 3.5 to 4.5 hours.

[0037] In a preferred technical solution of the present invention, the solvent for washing the cells in step S-1 is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, and the number of cell washing is 2 to 5 times, preferably 3 to 4 times.

[0038] In a preferred technical solution of the present invention, the separation described in step S-1 is any one selected from centrifugation and filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0039] In a preferred technical solution of the present invention, the ultrasonic conditions in step S-2 are 2°C to 8°C, 25 kHz, and 360 W, and ultrasonic treatment is performed for 1 to 5 minutes with an interval of 1 second for 3 seconds.

[0040] In a preferred technical solution of the present invention, the separation described in step S-3 is any one or combination selected from 2000-8000 rpm*10-30 min centrifugation, multi-stage centrifugation, and multi-stage filtration, preferably 3000-7000 rpm*15-25 min.

[0041] In a preferred technical solution of the present invention, the multi-stage centrifugation in step S-3 is performed in the order of 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0042] In a preferred technical solution of the present invention, the pore size of the filtration membrane of the multi-stage filtration is any one selected from 80 um, 50 um, 30 um, 10 um, and 5 um.

[0043] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is frozen and stored, preferably at -40°C to -20°C.

[0044] In a preferred technical solution of the present invention, the cell protein extract prepared in step S-3 is enzymatically digested using either nuclease or ultranuclease, and then separated and purified.

[0045] In a preferred technical solution of the present invention, the culture of the passaged mesenchymal stem cells or the culture of the primary mesenchymal stem cells is carried out by a culture method known in the art.

[0046] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing 5.0×10 primary mesenchymal stem cells. 5 ~5.0×10 6 The method includes the steps of adding the cells to a subculture medium, which is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, so as to achieve an initial density of cells / ml, and then culturing the medium at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days, and replacing half of the subculture medium every 2 to 3 days after observing the yellowing of the subculture medium.

[0047] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured by: After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was taken and cut into 3 mm pieces. 3 Then, the tissue pieces are cut into small pieces, centrifuged and washed to collect the tissue pieces, which are then 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℃ and 5%±1.0% CO2, changing half of the medium every 2-3 days, until cells emerge from the tissue pieces. and 2) shaking the mixture to collect the lower layer cells and washing them with PBS, then adding 0.25% trypsin to digest for 2-3 minutes, adding an equal volume of trypsin stop solution to stop the digestion, gently blowing with a pipette, centrifuging at 1200-1500 rpm / min for 5-8 minutes, and collecting the cells.

[0048] An object of the present invention is to provide a method for producing a neural repair protein composition, the method comprising: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting mixture to enzymatic degradation at 37°C±1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) adjusting the enzymatic degradation solution produced in step (1) to 5 to 15 mg / ml at 2°C to 8°C with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride, and then passing the solution through a chromatography column to monitor and collect the eluted fraction having an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0049] In a preferred technical solution of the present invention, the nuclease is selected from the group consisting of RNA nuclease, DNA nuclease, or a combination thereof.

[0050] In a preferred technical solution of the present invention, 25 U / mL to 30 U / mL of any one or a combination of nuclease or ultranuclease is added to the cell protein extract of the present invention, and the mixture is subjected to enzymatic hydrolysis at 37°C±1°C for 20 to 30 minutes to prepare an enzymatic hydrolysis solution.

[0051] 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.

[0052] In a preferred technical solution of the present invention, the protein composition obtained in step (2) is frozen and stored, preferably at -40°C to -20°C.

[0053] In a preferred technical solution of the present invention, the protein composition collected in step (2) is added with a lyoprotectant, which is any one or combination of selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch, and then lyophilized to prepare a lyophilized formulation of the protein composition.

[0054] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass.

[0055] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the protein composition collected in step (2), which is any one selected from albumin, zinc salts, and aluminum salts.

[0056] In a preferred technical solution of the present invention, the lyophilized preparation has a pH of 6-8, preferably 7-7.5.

[0057] In a preferred technical solution of the present invention, the protein composition in the nerve repair protein composition is as shown in FIG.

[0058] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted with saline or 5% glucose solution just before use, and then administered by intravenous injection, intrathecal injection, lumbar puncture or any combination thereof.

[0059] Another object of the present invention is to provide a neuro-repair composition comprising any one or combination of the cellular protein extracts or neuro-repair protein compositions having neuro-effects of the present invention and a pharma- ceutically acceptable carrier.

[0060] The dosage or type of the pharma- ceutically acceptable carrier of the present invention is determined by factors such as the physicochemical properties and content of the active ingredient in the composition, the type of formulation, the dissolution and bioavailability of the formulation, and the like.

[0061] The compositions of the present invention may be in any dosage form known in the art and may be prepared using formulation techniques known in the art.

[0062] In a preferred technical solution of the present invention, the composition is any one selected from the group consisting of freeze-dried agents, gelling agents, nasal sprays, liquid dressings, injections, patches, ointments, creams, emulsions, and suppositories.

[0063] In a preferred technical solution of the present invention, the administration method of the composition is selected from the following: intravenous injection, intrathecal injection, lumbar puncture, or a combination thereof.

[0064] Another object of the present invention is to provide a use of the cellular protein extract, the neural repair protein composition or the composition thereof having the neural cell repair effect of the present invention in the production of any one of products for cell repair or neural repair.

[0065] In a preferred technical solution of the present invention, the nerve repair is repair of nerve damage caused by any one of symptoms selected from central nervous system injury, neurodegenerative disease, stroke, brain injury, ataxia, intracerebral hemorrhage, Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, peripheral nerve injury, diabetes, and memory loss, or complications thereof.

[0066] In a preferred embodiment of the present invention, the ataxia is selected from the group consisting of cerebellar ataxia, cerebral ataxia, sensory ataxia, and vestibular ataxia, or a complication thereof.

[0067] Another object of the present invention is to provide a use of compounds 1-16 in stress-inducing stem cells to produce functional proteins with repair effects.

[0068] In a preferred technical solution of the present invention, the repair is any one selected from cell repair, hair follicle repair, joint repair, and nerve repair.

[0069] Unless otherwise stated, when the invention relates to percentages between liquids and liquids, said percentages are volume / volume percentages, when the invention relates to percentages between liquids and solids, said percentages are volume / weight percentages, when the invention relates to percentages between solids and liquids, said percentages are weight / volume percentages and the remainder are weight / weight percentages.

[0070] Unless otherwise specified, the mesenchymal stem cells (MSCs) of the present invention can be identified in accordance with "Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application."

[0071] The present invention detects reactive oxygen species (IOD), superoxide dismutase (SOD), and malondialdehyde (MDA) levels using commercially available kits. Effect of the Invention

[0072] Compared with the conventional technology, the present invention has the following advantageous effects:

[0073] 1. In the present invention, a medium containing a stress substance that induces mesenchymal stem cells to produce functional proteins with cell repair and nerve repair effects is scientifically screened. The obtained cell protein extract, protein composition or composition thereof has the effects of cell repair and nerve damage repair, and can be used to repair nerve damage caused by any one of the following symptoms or complications: central nervous system injury, neurodegenerative disease, stroke, brain injury, ataxia, cerebral hemorrhage, Alzheimer's disease, Parkinson's disease, dementia, multiple sclerosis, Huntington's disease, amyotrophic lateral sclerosis, peripheral nerve injury, diabetes, memory loss, cerebellar ataxia, cerebral ataxia, sensory ataxia, and vestibular ataxia, and has the advantages of high purity, good stability, high bioavailability, safety and effectiveness, and easy production, storage and transportation.

[0074] 2. The preparation method of the present invention has the advantages of being simple in operation, environmentally friendly, cost-effective, and suitable for industrial production. [Brief description of the drawings]

[0075] [Figure 1] Electrophoretic separation results of the nerve repair protein composition of the present invention. [Diagram 2] 1 shows the results of high performance liquid chromatography detection of the nerve repair protein composition of the present invention. [Diagram 3] This is a study of the repair effect of the cell protein extract of the present invention on skin damage caused by UVB radiation. [Figure 4]The effect of the cell protein extract of the present invention on the production of reactive oxygen species (IOD) in skin damage caused by UVB irradiation. Mean ± SD (n=3). ## indicates p<0.01 compared to the blank control (BC) group, * indicates p<0.05 compared to the NC (negative control) group, and ** indicates p<0.01 compared to the NC (negative control) group. [Diagram 5] The 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 to the blank control (BC) group, * indicates p<0.05 compared to the NC (negative control) group, and ** indicates p<0.01 compared to the NC (negative control) group. [Figure 6] The effect of the cell protein extract of the present invention on MDA level in skin damage caused by UVB irradiation. Mean ± SD (n=3). ## indicates p<0.01 compared to the blank control (BC) group, * indicates p<0.05 compared to the NC (negative control) group, and ** indicates p<0.01 compared to the NC (negative control) group. [Figure 7] 1 shows the results of comparing weekly body weights between a treatment group and a model group in a study of the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke. [Figure 8] 1 shows the results of TTC staining of a treatment group and a model group in a study of the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke. [Figure 9] 1 shows the results of immunofluorescence staining of iba1 positive cells in a treatment group and a model group in a study of the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke. [Figure 10] 1 shows the results of immunofluorescence staining of β3Tubulin-positive cells in a treatment group and a model group in a study of the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke. [Figure 11]1 shows the neuronal function (NDS) score results in a study of the damage repair effect of the neuronal repair protein composition of the present invention on ischemic stroke. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0076] The present invention will be further explained and described in detail with reference to the following specific examples, but the scope of protection of the present invention is not limited thereto.

[0077] 1. Cultivation of primary mesenchymal stem cells Primary mesenchymal stem cell culture After cleaning and disinfecting the umbilical cord, the tissue was dissected and the Wharton's jelly layer tissue was removed and cut into 3 mm pieces. 3 Then, cut the tissue into small pieces, centrifuge and wash to collect the tissue pieces, place them in a culture flask, add DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and then culture it under 37℃ and 5% CO2 conditions to promote adhesion to the wall. After observing the yellowing of the medium every 2-3 days, replace half of the medium and culture for 10-12 days until cells are seen emerging from the edge of the tissue pieces. 2) gently shaking the plate to remove the tissue fragments, and then collecting the tissue fragments and the lower layer cells, and then reattaching and culturing the collected tissue fragments; The collected lower layer cells are washed with PBS, and then an appropriate amount of 0.25% trypsin is added to digest the cells for 2 to 3 minutes. The digestion is stopped by adding an equal volume of trypsin stop solution. The bottom of the flask is gently blown with a pipette, and the cells are centrifuged at 1500 rpm / min for 5 minutes. Then, the cells are collected and obtained.

[0078] 2. Subculture of primary mesenchymal stem cells (culture of subcultured mesenchymal stem cells) Subculture of primary mesenchymal stem cells (subculture of mesenchymal stem cells): Primary mesenchymal stem cells were cultured at 5.0 x 10 5 ~5.0×10 6Add the cells to DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin to an initial density of cells / ml, then culture it at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 to 15 days. After observing the yellowing of the medium every 2 to 3 days, replace half of the medium.

[0079] 3. For the preparation of compounds 1 to 16, refer to Reference 1 (New limonophyllines AC from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch. Pharm. Res. (2018) 41:431-437).

[0080] Example 1: Preparation of cell protein extract having nerve repair effect according to the present invention The method for producing a cell protein extract having a nerve repair effect of the present invention comprises the steps of: Mesenchymal cell lineages: 8.0 x 10 6 To obtain a density of 1000 cells / mL, add the medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 5μmol / L compound 16, then culture it at 37℃, 5% CO2 for 4 hours, then centrifuge it at 1200rpm*5min, wash it with PBS three times, and collect the cells (1); The cells collected in step (1) were diluted to 1.0 × 10 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell protein extract. If necessary, the cell protein extract can be frozen and stored at -40°C to -20°C.

[0081] Example 2: Preparation of the nerve repair protein composition of the present invention The preparation of the neural repair protein composition of the present invention includes the steps of: (1) adding 25 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 1, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic hydrolysis solution prepared in step (1) at 10 mg / ml with an elution solvent containing 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8) at 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, and monitoring and collecting the eluted fraction having an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm.

[0082] Example 3: Preparation of a freeze-dried formulation of the nerve repair protein composition of the present invention Mannitol was added to the cell protein composition prepared in Example 2, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 2.15% mannitol (m / m).

[0083] Example 4: Preparation of cell protein extracts having nerve repair effects according to the present invention The method for producing a cell protein extract having a nerve repair effect of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7To obtain a density of 1000 cells / mL, add the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 8μmol / L compound 13, then culture the medium at 37℃ and 5% CO2 for 4 hours, then centrifuge the medium at 1200rpm*5min, wash the medium with PBS three times, and then collect the cells (1); 2.0 × 10 cells collected in step (1) 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0084] Example 5: Preparation of the nerve repair protein composition of the present invention The preparation of the neural repair protein composition of the present invention includes the steps of: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 4, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic digestion solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in sequence at an elution flow rate of 0.5 ml / min and monitoring and collecting the protein composition at an ultraviolet wavelength of 280 nm to obtain the protein composition.

[0085] Example 6: Preparation of a freeze-dried formulation of the nerve repair protein composition of the present invention Dextran was added to the cell protein composition prepared in Example 5, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 1% dextran (m / m).

[0086] (Example 7) Preparation of cell protein extract having nerve repair effect of the present invention The method for producing a cell protein extract having a nerve repair effect of the present invention comprises the steps of: Mesenchymal subculture cells 9.0 x 10 6 To obtain a density of 1000 cells / mL, add the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 5μmol / L compound 14, then culture the medium at 37℃ and 5%CO2 for 4h, then centrifuge the medium at 1200rpm*5min, wash the medium with PBS three times, and then collect the cells (1); 2.0 × 10 cells collected in step (1) 7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0087] Example 8: Preparation of the nerve repair protein composition of the present invention The preparation of the neural repair protein composition of the present invention includes the steps of: (1) adding 20 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 7, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic digestion solution prepared in step (1) at 10 mg / ml with an elution solvent containing 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8) at 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in that order, at an elution flow rate of 0.1 to 1 ml / min, and monitoring and collecting the protein composition at an ultraviolet wavelength of 280 nm to obtain the protein composition.

[0088] Example 9: Preparation of a freeze-dried formulation of the nerve repair protein composition of the present invention Sorbitol was added to the cell protein composition prepared in Example 8, stirred and mixed uniformly, and then lyophilized to obtain a lyophilized formulation containing 5% sorbitol (m / m).

[0089] (Example 10) Preparation of cell protein extract having nerve repair effect of the present invention The method for producing a cell protein extract having a nerve repair effect of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7 To obtain a density of 1000 cells / mL, add the cells to a medium containing 45% DMEM / F12, 45% RPMI1640, 0.5% bovine serum albumin (BSA), 10ug / mL epidermal growth factor (EGF), 10ug / mL fibroblast growth factor (FGF), 10ug / mL insulin transferrin, 0.05% amino acid compound (18AA) and 6μmol / L compound 15, then culture the medium at 37℃ and 5% CO2 for 4 hours, then centrifuge the medium at 1200rpm*5min, wash the medium with PBS three times, and then collect the cells (1); 2.0 × 10 cells collected in step (1)7 (2) dispersing the cells in physiological saline to a density of 1000 / mL, and sonicating for 2 min at 2-8 °C, 25 kHz, and 360 W with an interval of 1 s for 3 s to prepare a cell lysate; and (3) centrifuging the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filtering the resulting centrifuged solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate.

[0090] Example 11: Preparation of the nerve repair protein composition of the present invention The preparation of the neural repair protein composition of the present invention includes the steps of: (1) adding 30 U / mL of ultranuclease (UCF.ME UltraNuclease) to the cell protein extract prepared in Example 10, and carrying out enzymatic digestion at 37° C. for 30 minutes to prepare an enzymatic digestion solution; and (2) preparing the enzymatic digestion solution prepared in step (1) at 10 mg / ml with an elution solvent containing 50 mmol / L phosphate buffer (pH 6.8) and 300 mmol / L sodium chloride at 2°C to 8°C, passing the solution through a high-purity silica gel liquid chromatography guard column (WondaGuard C18, 4.6 x 5 mm) and a high-purity silica gel liquid chromatography preparation column (SHIMSEN Ankylo C18, 5 μm, 4.6 x 250 mm) in sequence at an elution flow rate of 1 ml / min and monitoring and collecting the protein composition at an ultraviolet wavelength of 280 nm to obtain the protein composition.

[0091] (Example 12) Preparation of a freeze-dried preparation of the nerve repair protein composition of the present invention Mannitol was added to the cell protein composition prepared in Example 11, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 3% mannitol (m / m).

[0092] (Example 13) Preparation of freeze-dried preparation of cell protein extract of the present invention A required amount of mannitol was added to the cell protein extract prepared in Example 1, stirred and mixed uniformly, and then lyophilized, so that the resulting lyophilized preparation contained 5% mannitol (m / m).

[0093] Example 14: Detection of molecular weight distribution of the nerve repair protein composition of the present invention The molecular weight distribution of the nerve repair protein composition of the present invention was detected using standard molecular weight polyacrylamide gel electrophoresis; Step 1: select a glass plate with a thickness of 1.5 mm, place it horizontally, and apply the prepared gel solutions (15% lower gel, 4% separating gel) on the glass plate in sequence according to Table 1, and insert the comb vertically into the lower gel; [Table 2] After thawing the marker (20KDa-245KDa) frozen at -40°C, the lyophilized powder of the marker and the nerve repair protein composition of Example 3, Example 6, Example 9, and Example 12 are mixed with PBS to prepare a test sample of 10ug / ul, and 20ul of the test sample is added to the loading well, and then electrophoresis is performed under 60-80V conditions until a relatively clear band appears, and the voltage is adjusted to 100-120V until marker separation is completed, and the PAGE gel is placed in Coomassie Brilliant Blue staining solution, and when a clear band appears on the gel, the staining is stopped, and after washing with pure water, it is destained with a 10% acetic acid solution, and destaining is stopped when the gel becomes transparent. The results are shown in FIG. 1. Here, band A is Example 3, band B is Example 6, band C is Example 9, and band D is Example 12.

[0094] Example 15: High performance liquid chromatography detection of the nerve repair protein composition of the present invention The lyophilized powder of the nerve repair protein composition of Example 3 was dissolved in deionized water and then formulated into a test solution of 10 mg / ml.

[0095] Chromatography column: SHIMSEN Ankylo (300mm*4.6mm.D., 3um; P / N: 380-01215-05) Shimadzu Mobile phase: 50 mmol / L phosphate buffer (pH = 6.8) containing 300 mmol / L sodium chloride, flow rate: 0.3 mL / min, injection volume: 10 ul, column temperature: 25°C, detection wavelength: 280 nm, isocratic elution, 30 min collection. See Figure 2 for the results. (Test Example 1) Study of the repair effect of the cell protein extract of the present invention on skin damaged by UVB radiation 3D Skin Model: TIFF2025504071000005.tif5164, commercially purchased from EpiKutis.

[0096] Model culture medium: DMEM basal medium.

[0097] VE working solution: 0.5 g of VE stock solution was aspirated and dissolved in 10 mL absolute ethanol to prepare a 5% mother solution, and 100 μL of the 5% mother solution was aspirated and dissolved in 10 mL model culture medium to prepare a 0.05% VE working solution.

[0098] 0.9 mL model culture medium was added to a 6-well plate, and the 3D skin model was transferred to the 6-well plate and labeled with the test number.

[0099] Blank control (BC): The skin model was not subjected to any treatment and was incubated in a CO2 incubator (37°C, 5% CO2) for 48 hours.

[0100] Negative control (NC): A dose of 600 mJ / cm on the surface of the skin model 2 After irradiation with UVB, the plates were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h. 25 μL of 0.2% SLS working solution was added, and the plates were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h.

[0101] Positive control (PC): A dose of 600 mJ / cm on the surface of the skin model 2 After irradiation with UVB, the cells were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours. 25 ul of 0.05% VE working solution was added, and the cells were then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0102] Test group: Dose of 600 mJ / cm on the surface of the skin model 2 After irradiation with UVB, the cells were placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours. 25 ul of a 1% cell protein extract solution (a 1% solution of the freeze-dried powder of the cell protein extract prepared in Example 13 with saline) was added, and the cells were then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0103] After 48 hours of incubation, the skin model was washed with PBS solution to remove any remaining liquid inside or outside the skin model. After 24 hours of fixation with 4% paraformaldehyde, the model was removed by circular excision and observed after H&E staining, the results of which are shown in Figure 3. The levels of reactive oxygen species (ROS), superoxide dismutase, and malondialdehyde (MDA) in the skin damaged by UVB radiation were detected, the results of which are shown in Figures 4 to 6. The cell protein extract of the present invention has obvious repairing effects on skin damaged by UVB radiation. (Test Example 2) Consideration of the repair effect of the nerve repair protein composition of the present invention on damage caused by ischemic stroke Seventy-five SPF male SD rats aged 6 to 7 weeks were selected. After the test animals were adapted to normal diet for one week, all animals underwent cerebral ischemia-reperfusion surgery to establish a stroke model. After the reperfusion of the test animals was completed, the neurological function was immediately evaluated when the test animals woke up from anesthesia.

[0104] The rats were fasted overnight on the day before the experiment, anesthetized, and fixed on a surgical board in a supine position. The neck was incised vertically about 3 cm to expose the common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA), and bluntly dissected. A slip knot was tied at the end of the common carotid artery (CCA) close to the heart to temporarily block the blood supply. A double knot was tied in the external carotid artery (ECA). A small opening was made in the middle of the double knot with microscissors to insert a wire plug. The external carotid artery (ECA) was cut from the middle of the double knot and the direction of the wire plug was adjusted so that it would enter the internal carotid artery (ICA). Using the bifurcation of the internal carotid artery (ICA) and external carotid artery (ECA) as the reference position, the wire plug was inserted up to the preset scale. When resistance was felt, it indicated that the head of the wire plug had already reached the middle cerebral artery (MCA), and a knot was tied to fix the wire plug. Ischemia was completed for 60 min by removing the blood clot and tying the common carotid artery slip knot.

[0105] 62 test animals survived after surgery. According to the scoring results, the test animals were divided into a model group (30 animals) and a treatment group (32 animals), and each was injected into the lateral ventricle three times, including administration immediately after cerebral ischemia-reperfusion, administration one week after surgery, and administration for two weeks after surgery. Corresponding to the treatment group (the lyophilized powder of the nerve repair protein composition of Example 3 was dissolved in saline, the dosage was 6.77 mgf / kg, and the administration volume was 20 ul), the model group was administered the same volume of saline into the lateral ventricle. Three days after surgery, two animals were selected from each group and TTC stained; four weeks after surgery, 15 animals were selected from the model group and 16 animals were selected from the treatment group, and one test animal was selected from each group and TTC stained; eight weeks after surgery, 13 animals were selected from the model group and 14 animals were selected from the treatment group, and one test animal was selected from each group and TTC stained.

[0106] During the test period, the test animals were observed once a week, including mortality, mental state, behavioral activity, onset, respiration, secretions, feces, food and water intake. The body weight of all test animals was detected once a week, and the results are shown in Figure 7. During the test period, there was no obvious difference in body weight between the treatment group and the model group.

[0107] The consistency of the stroke model was evaluated 3 days after surgery, 4 weeks after surgery, and 8 weeks after surgery. After blood collection, the left ventricle was immediately cannulated, and physiological saline was rapidly poured in to rinse for 2 minutes. The head was cut to remove the brain, and TTC staining was performed. The brain was cut into 5 to 7 coronal slices from front to back, and the slice thickness was about 1.8 mm. After staining with 1% TTC at 37 °C for 10 minutes, it was fixed with tissue fixative, and then scanned using a scanner and analyzed with analysis software. When it reacts with dehydrogenase in normal tissue, it shows red color, but because the activity of dehydrogenase in ischemic tissue is reduced and it cannot react, it remains unchanged and shows pale color. The results are shown in Figure 8.

[0108] The animal's limb grip strength was measured and evaluated before and one week after surgery using an animal limb grip strength measuring device. A rotational fatigue tester was used to measure the whole body endurance of the test animals before and one week after surgery, with a rotational speed of 12r / min, and the experiment was performed three times in a row, each time for 3 minutes, including the average values ​​of the three tests for the speed at the time of falling, the running duration experiment, and the running distance, and the results are shown in Table 2. The grip strength of the forelimbs, the speed before the fall, the running distance, and the duration were all higher in the treatment group than in the model group one week after surgery (P<0.01).

[0109] [Table 3] Note: Compared to the model group, * indicates P<0.05 and ** indicates p<0.01.

[0110] The animals were tested for learning and memory function in the water maze test 4 weeks and 8 weeks after surgery, and their activity was evaluated. The acquisition training experiment was conducted first for the first 4 days, and the platform exploration experiment was conducted on the 5th day. Acquisition training experiment method: First, the platform was exposed to the water surface so that the rat could see the platform. Next, the rat was placed in the pool, and if the rat swam directly toward the platform without any problems, it was explained that its swimming ability and vision were both normal, and the experiment could be started. The platform was attached to a position 5 mm below the water surface in the second quadrant, and the rat was placed in the water with its back to the wall of the pool. The position for the placement was chosen from the starting positions of the four quadrants 1, 2, 3, and 4, which were random and did not overlap, and the rat was placed in each of the four quadrants once, for a total of four times, and the time it took for the rat to find the platform under the water (escape latency period) was recorded. When the escape latency period exceeded 60 s, the rat was guided to the platform, and after staying on the platform for 10 s, the rat was moved and wiped. If necessary, the rats were exposed to a 150W incandescent lamp for 5 minutes and then returned to their cages. Platform exploration experiment method: After the acquisition training was completed, the platform in the second quadrant was removed the next day, and the rats were placed in the water at the starting position of the fourth quadrant, with their heads facing the wall of the pool. After the experiment was completed, the number of times the rats crossed the platform in the acquisition experiment was analyzed, and the results are shown in Table 3. The number of times the rats crossed the platform 4 weeks and 8 weeks after surgery was higher in the treatment group than in the model group (P<0.01).

[0111] [Table 4] Note: Compared to the model group, * indicates P<0.05 and ** indicates p<0.01.

[0112] Pathological detection: 3mm thick tissue samples were collected, dehydrated stepwise in 70%, 80%, 95%, and 100% alcohol for 30min each, treated with two xylene bottles for 20min each, embedded in paraffin in two wide-mouth jars for 30min each, cut into 4-micron slices, baked, and stained. The results are shown in Tables 4 and 5. Eight weeks after surgery, the atrophy percentage and calcification area of ​​the healthy side relative to the injured side were both significantly lower in the treatment group than in the model group (P<0.01).

[0113] [Table 5] Note: Compared to the model group, * indicates P<0.05 and ** indicates p<0.01.

[0114] [Table 6] Note: Compared to the model group, * indicates P<0.05 and ** indicates p<0.01.

[0115] For the results of immunofluorescence staining, see Table 6 and Figures 9 to 10. The area of ​​iba1 positive cells 4 weeks and 8 weeks after surgery was significantly lower in the treatment group than in the model group (p<0.05), and the number of β3 Tubulin positive cells 8 weeks after surgery was significantly higher in the treatment group than in the model group (p<0.01).

[0116] [Table 7] Note: Compared to the model group, * indicates P<0.05 and ** indicates p<0.01. (Test Example 3) Study of the damage repair effect of the nerve repair protein composition of the present invention on ischemic stroke Eighty SPF SD male rats weighing 220-240g were selected. The test animals were allowed to eat and drink ad libitum.

[0117] The test animals were anesthetized with isoflurane gas and maintained in anesthesia. The animals were fixed in a supine position, the skin was incised along the midline of the neck, the right common carotid artery was exposed, and the nerves and fascia branching around the common carotid artery at the base of the skull were carefully separated. The occipital artery, superior thyroid artery, lingual artery, and maxillary artery branches of the external carotid artery were isolated, ligated, and cut. The external carotid artery was separated from the end far from the heart by 3 mm. # A nylon thread (0.25 mm in diameter) was introduced into the internal carotid artery and inserted into the middle cerebral artery (MCA) at the circle of Willis to effectively block the MCA, and the length of the inserted nylon thread was 18-20 mm from the common carotid artery bifurcation point. Two hours after MCAO, the nylon thread in the internal carotid artery vascular lumen was carefully pulled out to reperfuse the internal carotid artery, and after the animals were fully awakened, the neurological function was scored (NDS, see Table 7). If the test animals scored >8 points, the modeling was successful.

[0118] [Table 8] Sixty rats that were successfully modeled were selected and randomly divided into six groups of 10 rats each according to the score, and the administration method was as shown in Table 8. After reperfusion, the test animals were administered intravenously.

[0119] [Table 9] The test animals were scored for their neurological function using a blind test before administration, and on the first, third, and fifth days after administration. The test data were shown as mean ± standard deviation (Mean ± SEM), and statistical analysis was performed using SPSS 25.0 software. If the variances matched, one-way analysis of variance was performed, and if there was a significant difference, the LSD test was used to compare pairs by pairs. If the variances did not match, the Kruskal-Wallis nonparametric test was used to compare pairs by pairs. The results are shown in Figure 11. Compared with before administration, test group 1 showed a comparable stroke neurological damage repair effect to positive control 1 and positive control 2 at 5 d after administration (P<0.05); compared with before administration, test group 2 showed a significant neurological damage repair effect at 3 d after administration (P<0.05); compared with before administration, test group 2 showed a more significant stroke neurological damage repair effect than positive control 1 and positive control 2 at 5 d after administration (P<0.01).

[0120] The above description of the specific embodiments of the present invention is not intended to limit the present invention, and those skilled in the art may make various modifications or variations based on the present invention, and any such modifications or variations should fall within the scope of protection of the claims of the present invention as long as they do not deviate from the spirit of the present invention.

Claims

1. A neural repair protein composition, the preparation of which comprises: (1) adding 20 U / mL to 35 U / mL of any one or a combination of nuclease and ultranuclease to the cell protein extract of the present invention, and subjecting the resulting mixture to enzymatic degradation at 37°C ± 1°C for 15 to 40 minutes to prepare an enzymatic degradation solution; and (2) preparing the enzymatic decomposition solution prepared in step (1) at 5 to 15 mg / ml with an elution solvent containing 300 mmol / L sodium chloride in 50 mmol / L phosphate buffer (pH 6.8) at 2°C to 8°C, passing the solution through a chromatography column, and monitoring and collecting an elution fraction having an elution flow rate of 0.1 to 1 ml / min and an ultraviolet wavelength of 280 nm. Neural repair protein compositions.

2. The preparation of the protein extract comprises: Density is 5.0 x 10 ６ pieces / mL ~ 1.0×10 ７ The mesenchymal stem cells / mL were placed in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum protein (BSA), 1-15 ug / mL epidermal growth factor (EGF), 1-15 ug / mL fibroblast growth factor (FGF), 1-15 ug / mL insulin transferrin, 0.01-0.1% amino acid compound (18AA), and 2-10 μmol / L of any one or combination of compounds 1-16, and then incubated at 37.0° C.±0.5° C., 5%±1.0% CO ２ Step S-1 of culturing the cells for 2 to 6 hours under the above conditions, and then isolating, washing, and collecting the cells; 【Table 1-1】 【Table 1-2】 The collected cells were 5.0 × 10 ６ pieces / mL ~ 5.0×10 ７ Step S-2 of dispersing the cells in a solvent selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer to a density of 10 ... Step S-3: separating the cell lysate prepared in step S-2, and then filtering the resulting separation solution through 0.45 um and 0.22 um filtration membranes in sequence to obtain a cell lysate. The protein composition of claim 1.

3. The medium in step S-1 contains 42-45% DMEM / F12, 42-45% RPMI1640, 0.5-1.5% bovine serum albumin (BSA), 5-10 ug / mL epidermal growth factor (EGF), 5-10 ug / mL fibroblast growth factor (FGF), 5-10 ug / mL insulin transferrin, 0.02-0.05% amino acid compound (18AA), and 3-8 μmol / L stress substance. A protein composition according to any one of claims 1 to 2.

4. The medium for step S-1 contains 45% DMEM / F12, 45% RPMI1640, 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% amino acid compound (18AA), and 4 to 6 μmol / L of a stress substance. A protein composition according to any one of claims 1 to 3.

5. The molecular weight of the nerve repair protein composition is 20 kDa to 250 kDa, preferably 35 kDa to 200 kDa; A protein composition according to any one of claims 1 to 4.

6. Add a lyoprotectant selected from mannitol, sorbitol, dextran, trehalose, glycerol, sucrose, glucose, lactose, maltose, glucan, tricaprylin, polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch, or a combination thereof, to the protein composition prepared in step (2), and lyophilize the mixture to prepare a lyophilized preparation of the protein composition. A protein composition according to any one of claims 1 to 5.

7. The freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.5 to 8%, preferably 1 to 5%, by mass. A protein composition according to any one of claims 1 to 6.

8. A nerve repair composition comprising the nerve repair protein composition of any one of claims 1 to 7 and a pharma- ceutically acceptable carrier.

9. Use of the nerve repair protein composition according to any one of claims 1 to 7 or the nerve repair composition according to claim 8 in the production of any one of the following products: cell repair, nerve repair.

10. Use of compounds 1-16 in stress-inducing stem cells to produce functional proteins having a repair effect.

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