Cell repair protein extract and its preparation and use

Abstract

The present invention relates to a cell protein extract having a cell repair effect, the preparation of which includes the following steps: Step (1): Extracting a cell protein extract having a density of 1.0×10 6 pieces / mL~5.0×10 7 Mesenchymal passaged cells per mL are placed 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% complex amino acid (18AA), and 2-10μmol / L stress substance, which is cultured at 37.0℃±0.5℃ and 5%±1.0% CO2 for 10min-14h, then separated, washed, and collected, where the stress substance is any one or a combination of compounds 1-16. Step (2): The collected cells are sonicated at 2℃-8℃ to produce a cell lysate, which is filtered to obtain. The obtained cell protein extract or composition thereof of the present invention can promote tissue endogenous repair, and can be used for repairing nerve damage, joint damage, damaged hair follicle cells, and skin damage, and has the advantages of high purity, good stability, safety, and effectiveness, effectively solving the problem that viable cells require refrigeration and their activity is limited by the cell viability time.

Description

[Technical field]

[0001] The present invention is in the field of biomedical technology, specifically, it relates to cell repair protein extracts and their preparation and use. [Background technology]

[0002] Cells are the basic units of life activity and the basis of the health of living organisms. When the redox balance of living organisms is destroyed, it will cause the interruption of redox signaling and control, which will induce oxidative stress disorders and lead to various diseases. Timely repair of damaged cells can significantly improve and treat related pathologies.

[0003] 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 produced by extraction from a citrus plant (Atalantia monophylla) have activity to inhibit the proliferation of tumor cells, etc.

[0004] Mesenchymal stem cells (MSCs) have the ability to self-renew and differentiate in multiple directions. They are widely distributed 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 supply 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) and other activating factors. They are involved in the regulation of processes such as cell proliferation, apoptosis, cell differentiation, antiviral and immune maturation, and 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 require refrigeration during production, storage, transportation, and use, and cell viability must be maintained for ≦12 h, limiting their therapeutic use. Therefore, there is a need to develop a safe and effective skin repair drug to meet clinical needs. Summary of the Invention

[0005] The object of the present invention is to provide a cell protein extract having a cell repair effect, the production of which comprises: Density is 1.0×10 6 pieces / mL~5.0×10 7 Step (1) of placing mesenchymal passaged cells / mL in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum albumin (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% complex amino acid (18AA) and 2-10 μmol / L stress substance, culturing the medium at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 min to 14 h, separating, washing and collecting the cells, wherein the stress substance is any one selected from compounds 1 to 16 or a combination thereof; [Table 1-1] [Table 1-2] The collected cells were diluted to 5.0 x 10 6 pieces / mL~1.0×10 7 Step (2) dispersing the cells in a solvent at a density of 10 ... and (3) separating the cell lysate produced in step (2), and then filtering the resulting separated solution through 0.45 um and 0.22 um filtration membranes, to obtain a cell lysate.

[0006] In a preferred technical solution of the present invention, the medium in step (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% complex amino acid (18AA), and 3-8 μmol / L stress substance.

[0007] In the preferred technical solution of the present invention, the medium in step (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% complex amino acid (18AA), and 4-6 μmol / L stress substance.

[0008] In a preferred technical solution of the present invention, the mesenchymal passage cell density in step (1) is 2.0×10 6 ~2.0×10 7 5.0×10 6 ~1.0×107 pieces / mL.

[0009] In a preferred technical solution of the present invention, the mesenchymal passaged cells in step (1) are cultured in the medium for 30 min to 13 h, preferably 45 min to 12 h.

[0010] In a preferred technical solution of the present invention, the solvent for washing the cells in step (1) is selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or any combination thereof, and the number of washing the cells is 2-5 times, preferably 3-4 times.

[0011] In a preferred technical solution of the present invention, the separation described in step (1) is any one selected from centrifugation, filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0012] In the preferred technical solution of the present invention, the ultrasonic conditions in step (2) are: 2°C to 8°C, 25 kHz, 360 W, operating for 3 s, with an interval of 1 s, and ultrasonic treatment for 1 to 5 min.

[0013] In a preferred technical solution of the present invention, the separation described in step (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.

[0014] In a preferred technical solution of the present invention, the multi-stage centrifugation in step (3) is, in order, 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0015] 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.

[0016] In the preferred technical solution of the present invention, the protein extract prepared in step (3) is frozen and stored, preferably at -40℃ to -20℃.

[0017] In a preferred technical solution of the present invention, the filtrate collected in step (3) is added with a lyoprotectant and then lyophilized to obtain the lyoprotectant, wherein the lyoprotectant is any one or combination selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch.

[0018] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.7 to 7%, preferably 1 to 5%, by mass.

[0019] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the filtrate collected in step (3), wherein the protein stabilizer is any one selected from albumin, zinc salt, and aluminum salt.

[0020] In a preferred technical solution of the present invention, the lyophilized preparation has a pH of 6-8, preferably a pH of 7-7.5.

[0021] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted with an isotonic solution just before use, and then used by any one or combination of the following methods: smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture, wherein the isotonic solution is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer.

[0022] 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 in the art.

[0023] In a preferred technical solution of the present invention, the culture of the passaged mesenchymal stem cells is carried out 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 at an initial density of 10 cells / ml, 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, wherein the subculture medium is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin.

[0024] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured: 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 Step 1) of cutting the tissue into small pieces, centrifuging, washing, collecting the tissue pieces, placing them in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and culturing them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, replacing half of the medium every 2-3 days, and culturing 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.

[0025] The object of the present invention is to provide a method for producing a cell protein extract having a cell repair effect, the method comprising the steps of: Density is 1.0×10 6 pieces / mL~5.0×10 7 Step (1) of placing mesenchymal passaged cells (cells / mL) in a medium containing 40-50% DMEM / F12, 40-50% RPMI1640, 0.1-2% bovine serum albumin (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% complex amino acid (18AA) and 2-10 μmol / L stress substance, culturing the medium at 37.0°C ± 0.5°C and 5% ± 1.0% CO2 for 10 min to 14 h, and then isolating, washing and collecting the cells, wherein the stress substance is any one selected from compounds 1 to 16 or a combination thereof; The collected cells were cultured at a density of 5.0 x 10 6 pieces / mL~1.0×10 7 Step (2) of dispersing the cells in a solvent at 100 μg / mL and subjecting the resultant to ultrasonic treatment at 2°C to 8°C to produce a cell lysate, wherein the solvent is any one selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer, or a combination thereof; and (3) separating the cell lysate produced in step (2), and then filtering the resulting separated solution through 0.45 um and 0.22 um filtration membranes, to obtain a cell lysate.

[0026] In a preferred technical solution of the present invention, the culture medium in step (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% complex amino acid (18AA), and 3-8 μmol / L stress substance.

[0027] In a preferred technical solution of the present invention, the culture medium in step (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% complex amino acid (18AA), and 4-6 μmol / L stress substance.

[0028] In a preferred technical solution of the present invention, the mesenchymal passage cell density in step (1) is 2.0×10 6 ~2.0×10 7 5.0×10 6 ~1.0×10 7 pieces / mL.

[0029] In a preferred technical solution of the present invention, the mesenchymal passaged cells in step (1) are cultured in the medium for 30 min to 13 h, preferably 45 min to 12 h.

[0030] In a preferred technical solution of the present invention, the solvent for washing the cells in step (1) is selected from physiological saline, 5% glucose solution, phosphate buffered saline (PBS), TBPS buffer, TBST buffer, and Tris buffer, or any combination thereof, and the number of washing the cells is 2-5 times, preferably 3-4 times.

[0031] In a preferred technical solution of the present invention, the separation described in step (1) is any one selected from centrifugation, filtration or a combination thereof, wherein the centrifugation conditions are 1000-2000 rpm*3-15 min, preferably 1200 rpm-1500 rpm*5-10 min.

[0032] 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 in the art.

[0033] In a preferred technical solution of the present invention, the mesenchymal stem cell culture is performed by culturing primary mesenchymal stem cells at an initial density of 5.0×10 5 ~5.0×10 6 The method includes the steps of adding the cells to a subculture medium at a concentration of 10 cells / ml, 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 subculture medium for yellowing, wherein the subculture medium is DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 ug / ml streptomycin.

[0034] In a preferred technical solution of the present invention, the primary mesenchymal stem cells are cultured: 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 Step 1) of cutting the tissue into small pieces, centrifuging, washing, collecting the tissue pieces, placing them in DMEM / F12 medium containing 10% fetal bovine serum (FBS), 100ug / ml penicillin, and 100ug / ml streptomycin, and culturing them under conditions of 37.0℃±0.5℃ and 5%±1.0% CO2, replacing half of the medium every 2-3 days, and culturing 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.

[0035] In the preferred technical solution of the present invention, the ultrasonic conditions in step (2) are: 2°C to 8°C, 25 kHz, 360 W, operating for 3 s, with an interval of 1 s, and ultrasonic treatment for 1 to 5 min.

[0036] In the preferred technical solution of the present invention, the separation in step (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.

[0037] In a preferred technical solution of the present invention, the multi-stage centrifugation in step (3) is, in order, 3000-4000 rpm*3-5 min, 5000-6000 rpm*3-5 min, and 7000 rpm*5-8 min.

[0038] 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.

[0039] In the preferred technical solution of the present invention, the protein extract obtained in step (3) is frozen and stored, preferably at -40℃ to -20℃.

[0040] In a preferred technical solution of the present invention, the filtrate collected in step (3) is added with a lyoprotectant and then lyophilized to obtain the lyoprotectant, wherein the lyoprotectant is any one or combination selected from mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch.

[0041] In a preferred technical solution of the present invention, the freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.7 to 7%, preferably 1 to 5%, by mass.

[0042] In a preferred technical solution of the present invention, a protein stabilizer is optionally added to the filtrate collected in step (3), wherein the protein stabilizer is any one selected from albumin, zinc salt, and aluminum salt.

[0043] In a preferred technical solution of the present invention, the lyophilized preparation has a pH of 6-8, preferably a pH of 7-7.5.

[0044] In a preferred technical solution of the present invention, the freeze-dried preparation is reconstituted with an isotonic solution just before use, and then used by any one or combination of smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture, wherein the isotonic solution is any one or combination selected from physiological saline, 5% glucose solution, phosphate buffer solution (PBS), TBPS buffer, TBST buffer, and Tris buffer.

[0045] Another object of the present invention is to provide a cell repair composition comprising the cell protein extract having the cell repair effect of the present invention and a pharma- ceutically acceptable carrier.

[0046] The amount 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.

[0047] The compositions of the present invention may be in a variety of dosage forms known in the art and can be prepared using formulation techniques known in the art.

[0048] 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, ointments, creams, emulsions, liquid dressings, injections, and suppositories.

[0049] In a preferred technical solution of the present invention, the administration method of the composition is selected from the following: smear, rolling needle, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupuncture injection, and lumbar puncture, or any combination thereof.

[0050] Another object of the present invention is to provide a use of the cell protein extract or composition thereof having the cell repair effect of the present invention in the manufacture of any one of drugs or products for cell repair, joint repair, cartilage repair, skin damaged cell repair, nerve damaged cell repair, organ damage repair, pulmonary fibrosis repair, liver damage repair, kidney damage repair, ovarian repair, anti-Crohn's disease, anti-sub-health, and anti-aging.

[0051] Another object of the present invention provides the use of compounds 1-16 in stress-inducing stem cells to produce functional proteins with repair effects.

[0052] In a preferred technical solution of the present invention, said repair is any one of cell repair, hair follicle repair, joint repair, and nerve repair.

[0053] 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.

[0054] Unless otherwise specified, in the present invention, detection is carried out using the following method.

[0055] 1. Identification of mesenchymal stem cells (MSCs): Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application 2. Detect the levels of reactive oxygen species (IOD), superoxide dismutase (SOD), and malondialdehyde (MDA) using commercially available kits. Effect of the Invention

[0056] Compared with the conventional technology, the present invention has the following advantageous effects:

[0057] 1. In the present invention, a culture medium containing a stress substance that induces mesenchymal stem cells to produce cell proteins with cell repair effects is scientifically screened. The obtained cell protein extract has the effects of repairing nerve damage, repairing joint damage, repairing damaged hair follicle cells, repairing skin damage, repairing pulmonary fibrosis, repairing liver damage, repairing kidney damage, repairing sub-health, repairing premature ovarian failure, preventing and treating Crohn's disease, and anti-aging, and has the advantages of high purity, good stability, safety and effectiveness, effectively solving the problem that viable cells need refrigeration and their activity is limited by the cell viability time.

[0058] 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]

[0059] [Figure 1] 1 is a study of the repair effect of the cellular protein extract of the present invention on oxidatively damaged skin. [Diagram 2] This is a study of the repair effect of the cell protein extract of the present invention on skin damage caused by UVB radiation. [Diagram 3] The effect of the cell protein extract of the present invention on the generation of oxidized oxide (IOD) in UVB-irradiated skin damage. 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 4]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. [Diagram 5] The effect of the cell protein extract of the present invention on MDA level in UVB-irradiated skin damage. Mean ± SD (n=3). ## indicates p<0.01 compared to blank control (BC) group, * indicates p<0.05 compared to NC (negative control) group, ** indicates p<0.01 compared to NC (negative control) group. [Figure 6] 13. Study of the repair effect of the protein extract of the present invention on medial condyle joint injury. [Figure 7] 1 is a study of the repair effect of the protein extract of the present invention on plateau joint injury. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] 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.

[0061] 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 Step 1) is to cut the tissue into small pieces, centrifuge, wash, 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 culture it under 37℃ and 5% CO2 conditions to promote attachment, and 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, 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.

[0062] 2. Subculture of primary mesenchymal stem cells Subculture of primary mesenchymal stem cells: 1.0×10 primary mesenchymal stem cells 5 -6.0×10 5 Add the cells at an initial density of cells / ml to DMEM / F12 medium containing 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin, and 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.

[0063] 3. For the production 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).

[0064] Example 1: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6The cells are added 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% complex amino acid (18AA) and 5μmol / L compound 16 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 30 minutes, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); The cells collected in step (1) were diluted to 8.0 × 10 6 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting them to ultrasonic treatment at 2 to 8 °C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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.

[0065] (Example 2) Production of freeze-dried preparation of cell protein extract of the present invention The required amount of mannitol was added to the cell protein extract prepared in Example 1, stirred and mixed uniformly, and then freeze-dried. The resulting freeze-dried preparation contained 2% mannitol (m / m).

[0066] Example 3: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages: 8.0 x 10 6The cells are added 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% complex amino acid (18AA) and 5μmol / L compound 16 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 4h, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); The cells collected in step (1) were diluted to 1.0 × 10 7 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting the cells to ultrasonic treatment at 2 to 8 ° C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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.

[0067] Example 4: Preparation of freeze-dried cell protein extract preparation of the present invention The required amount of mannitol was added to the cell protein extract prepared in Example 3, stirred and mixed uniformly, and then freeze-dried. The resulting freeze-dried preparation contained 5% mannitol (m / m).

[0068] Example 5: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6 The cells are added 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% complex amino acid (18AA) and 5μmol / L compound 16 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 12h, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); The cells collected in step (1) were diluted to 1.0 × 10 7 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting the cells to ultrasonic treatment at 2 to 8 ° C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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.

[0069] (Example 6) Preparation of freeze-dried cell protein extract preparation of the present invention The required amount of mannitol was added to the cell protein extract prepared in Example 5, stirred and mixed uniformly, and then freeze-dried. The resulting freeze-dried preparation contained 2% mannitol (m / m).

[0070] Example 7: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages: 5.0 x 10 6 The cells are added 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% complex amino acid (18AA) and 5μmol / L compound 13 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 30 minutes, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); 7.0 × 10 cells collected in step (1) 6 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting them to ultrasonic treatment at 2 to 8 °C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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 the cell protein extract. The cell protein extract produced can be freeze-dried as necessary.

[0071] (Example 8) Preparation of freeze-dried cell protein extract of the present invention Sorbitol was added to the cell protein extract prepared in Example 7, stirred and mixed uniformly, and then lyophilized to obtain a lyophilized preparation containing 3% sorbitol (m / m).

[0072] Example 9: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7 The cells are added 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% complex amino acid (18AA) and 8μmol / L compound 14 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 4h, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); 5.0 × 10 cells collected in step (1) 7 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting the cells to ultrasonic treatment at 2 to 8 ° C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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.

[0073] (Example 10) Preparation of freeze-dried preparation of cell protein extract of the present invention Dextran was added to the cell protein extract prepared in Example 9, stirred and mixed uniformly, and then lyophilized. The resulting lyophilized preparation contained 1% dextran (m / m).

[0074] Example 11: Preparation of the cell protein extract of the present invention The method for producing a cell protein extract of the present invention comprises the steps of: Mesenchymal cell lineages 1.0 x 10 7 The cells are added 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% complex amino acid (18AA) and 8μmol / L compound 15 at a density of 100 cells / mL, and then cultured at 37℃ and 5% CO2 for 12h, followed by centrifuging at 1200rpm*5min, washing three times with PBS, and then collecting the cells (1); 5.0 × 10 cells collected in step (1) 7 Step (2) of dispersing the cells in physiological saline at a density of 1000 / mL, subjecting the cells to ultrasonic treatment at 2 to 8 ° C, 25 kHz, and 360 W for 3 seconds at 1 second intervals, and sonicating for 2 minutes to produce a cell lysate; and (3) centrifuging the cell lysate produced 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.

[0075] (Example 12) Preparation of freeze-dried preparation of cell protein extract of the present invention Dextran was added to the cell protein extract prepared in Example 11, stirred and mixed uniformly, and then freeze-dried to obtain a freeze-dried preparation containing 2% dextran (m / m).

[0076] (Test Example 1) Study of the repair effect of the cell protein extract of the present invention on oxidatively damaged skin 3D Skin Model: TIFF2025503296000004.tif5164, commercially available EpiKutis purchased.

[0077] SLS working solution: 0.0080 g of SLS was weighed out and dissolved in 2 mL of PBS solution, and filtered through a 0.22 μm filter to prepare a 0.4% SLS mother solution. 0.5 mL of the 0.4% SLS mother solution was aspirated and 0.5 mL of PBS was added to prepare a 0.2% SLS working solution.

[0078] WY14643 working solution: 10 mg of WY14643 (PPARα stimulant) was weighed and dissolved in 1 mL of DMSO to prepare a 30 mM WY14643 mother solution. Then, 10 μL of the WY14643 mother solution (30 mM) was added to 6 mL of the model culture medium to prepare a 50 μM WY14643 working solution.

[0079] Model culture medium: DMEM basal medium.

[0080] 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.

[0081] Blank control (BC): No treatment was applied to the skin model, which was then incubated in a CO2 incubator (37°C, 5% CO2) for 48 h.

[0082] Negative control (NC): 25 μL of 0.2% SLS working solution was added to the surface of the skin model, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h.

[0083] Positive control (PC): 25 μL of 0.2% SLS working solution was added to the surface of the skin model and incubated in a CO2 incubator (37°C, 5% CO2) for 24 h, after which 25 μL of 50 μM WY14643 working solution was added and incubated in a CO2 incubator (37°C, 5% CO2) for 24 h.

[0084] Test group: 25 μL of 0.2% SLS working solution was added to the surface of the skin model, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours. Then, 25 μL of 1% cell protein extract solution (a 1% solution was prepared by adding the freeze-dried powder of the cell protein extract of Example 2 to saline) was added, which was then placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0085] After 48 hours of incubation, the skin model was washed with PBS solution to remove any remaining liquid inside and 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 can be seen in Figure 1. The protein extract of the present invention had obvious repairing effect on oxidatively damaged skin.

[0086] (Test Example 2) Study of the repair effect of the cell protein extract of the present invention on skin damaged by UVB radiation 3D Skin Model: TIFF2025503296000005.tif5164, commercially available EpiKutis purchased.

[0087] Model culture medium: DMEM basal medium.

[0088] VE working solution: 0.5 g of VE stock solution was aspirated and dissolved in 10 mL of absolute ethanol to prepare 5% mother solution, and 100 μL of the 5% mother solution was aspirated and dissolved in 10 mL of model culture medium to prepare 0.05% VE working solution.

[0089] 0.9 mL of 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.

[0090] Blank control (BC): No treatment was applied to the skin model, which was then incubated in a CO2 incubator (37°C, 5% CO2) for 48 h.

[0091] Negative control (NC): A dose of 600 mJ / cm on the surface of the skin model 2 After UVB irradiation, it was 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 it was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 h.

[0092] Positive control (PC): A dose of 600 mJ / cm on the surface of the skin model 2 After irradiating it with UVB, it was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h, 25 ul of 0.05% VE working solution was added, and it was placed in a CO2 incubator (37℃, 5% CO2) and incubated for 24 h.

[0093] Test group: Dose of 600 mJ / cm on the surface of the skin model 2 After irradiating with UVB, it was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours, and 25 ul of 1% cell protein extract solution (a 1% solution was prepared by adding the freeze-dried cell protein extract powder prepared in Example 4 of the present invention to saline) was added, and the mixture was placed in a CO2 incubator (37°C, 5% CO2) and incubated for 24 hours.

[0094] 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 can be seen in Figure 2. The levels of active oxygen (IOD), superoxide dismutase, and malondialdehyde (MDA) in the skin damaged by UVB radiation were detected, the results of which can be seen in Figures 3 to 5. The cell protein extract of the present invention had obvious repairing effects on the skin damaged by UVB radiation.

[0095] (Test Example 3) Study of the repair effect of the cell protein extract of the present invention on joint damage and cartilage damage A male goat, aged 12 months and weighing 47 kg, was generally bred under general anesthesia, and its left medial condyle was incised 8 cm along the midline, and the medial edge of the patellar tendon was incised up to the joint to expose the medial femoral condyle and the medial tibial plateau. The surface of the articular cartilage was found to be smooth, and a cartilage injury wound measuring 0.3×0.3×0.6 cm (length×width×depth) was made on the medial femoral condyle with a 3 mm ball drill, and the incision was sutured (see Figure 6). Its right lateral plateau was incised 8 cm along the midline, and separated layer by layer, and the joint capsule was entered on the lateral side of the patellar tendon to expose the lateral femoral condyle and the lateral tibial plateau. The cartilage was found to be intact and smooth, and a cartilage injury wound measuring 0.6×0.3×0.3 cm (length×width×depth) was made on the lateral side of the plateau with a 3 mm ball drill, and the incision was sutured (see Figure 7).

[0096] Goats were injected with 2.5 ml of the cell protein extract freeze-dried preparation solution of Example 6 (the cell protein extract freeze-dried preparation of Example 6 was prepared into a 2.4% solution with saline) into the knee joint cavity on one side every week for three consecutive weeks. Observations were performed once every morning and evening. The results can be seen in Table 1.

[0097] [Table 2] After 49 days of observation, the experimental goats were anesthetized and killed, and the material was taken to prepare cartilage slices (see Figures 6-7).

[0098] The cell protein extract of the present invention has an excellent effect of repairing joint damage, and is safe and effective.

[0099] 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 cell protein extract having a cell repair effect, the production of which comprises: Density is 1.0 x 10 ６ pieces / mL ~ 5.0×10 ７ The mesenchymal passaged 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% complex amino acid (18AA), and 2-10 μmol / L stress substance, and incubated at 37.0°C ± 0.5°C, 5% ± 1.0% CO ２ Step (1), in which the cells are cultured under the conditions for 10 min to 14 h, and then separated, washed, and collected, wherein the stress substance is any one selected from compounds 1 to 16 or a combination thereof; 【Table 1-1】 【Table 1-2】 The collected cells were diluted to 5.0 × 10 ６ pieces / mL ~ 1.0×10 ７ Step (2) dispersing the cells in a solvent at a density of 10 ... and (3) separating the cell lysate produced in step (2), and then filtering the resulting separated solution through 0.45 um and 0.22 um filtration membranes to obtain a cell lysate. A cell protein extract with cell repair effects.

2. The medium in step (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% complex amino acid (18AA), and 3-8 μmol / L stress substance; 2. The protein extract of claim 1.

3. The density of mesenchymal passage cells in step (1) was 2.0 × 10 ６ ~2.0 x 10 ７ 5.0×10 ６ ~1.0 x 10 ７ pcs / mL, A protein extract according to any one of claims 1 to 2.

4. The separation described in step (1) is any one selected from centrifugation and filtration, or a combination thereof, wherein the centrifugation conditions are 1000 to 2000 rpm * 3 to 15 min, preferably 1200 rpm to 1500 rpm * 5 to 10 min; A protein extract according to any one of claims 1 to 3.

5. The ultrasonic conditions in step (2) are 2°C to 8°C, 25 kHz, 360 W, 3 s operation, 1 s interval, 1 to 5 min ultrasonic treatment; A protein extract according to any one of claims 1 to 4.

6. adding a lyoprotectant to the filtrate collected in step (3) to produce a lyoprotectant formulation, wherein the lyoprotectant is any one or combination selected from the group consisting of mannitol, sorbitol, dextran, glycerol, sucrose, trehalose, glucose, lactose, maltose, glucan, tricaprylin (HES), polyethylene glycol, ethylenediamine, phosphate, acetate, citrate, sorbitol, and starch; A protein extract according to any one of claims 1 to 5.

7. The freeze-dried preparation contains a freeze-drying protection agent in an amount of 0.7 to 7%, preferably 1 to 5%, by mass. A protein extract according to any one of claims 1 to 6.

8. The lyophilized formulation has a pH of 6 to 8, preferably a pH of 7 to 7.

5. A protein extract according to any one of claims 1 to 7.

9. A cell repair composition comprising a cell protein extract having a cell repair effect according to any one of claims 1 to 8 and a pharma- ceutical acceptable carrier.

10. Use of a cell protein extract having cell repair effect described in any one of claims 1 to 8 or a composition described in claim 9 in the manufacture of any one of drugs or products for cell repair, joint repair, cartilage repair, skin damaged cell repair, nerve damaged cell repair, organ damage repair, pulmonary fibrosis repair, liver damage repair, kidney damage repair, ovarian repair, anti-Crohn's disease, anti-sub-health, and anti-aging.

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