WO2023143519A1 - 一种细胞修复蛋白提取物及其制备方法和其应用 - Google Patents

一种细胞修复蛋白提取物及其制备方法和其应用 Download PDF

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WO2023143519A1
WO2023143519A1 PCT/CN2023/073566 CN2023073566W WO2023143519A1 WO 2023143519 A1 WO2023143519 A1 WO 2023143519A1 CN 2023073566 W CN2023073566 W CN 2023073566W WO 2023143519 A1 WO2023143519 A1 WO 2023143519A1
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cells
cell
protein extract
repair
present
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PCT/CN2023/073566
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French (fr)
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王宇
高文勇
陈琳
李建军
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北京达尔文细胞生物科技有限公司
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Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a cell repair protein extract, a preparation method thereof and an application thereof.
  • Cells are the basic unit of life activities and the foundation of body health. When the body's redox balance is disrupted, redox signaling and control will be disrupted, leading to oxidative stress damage and various diseases. Timely repair of damaged cells can significantly improve and treat related lesions.
  • Mesenchymal stem cells have the potential of self-replication and multi-directional differentiation, 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, which have a wide range of sources, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, convenient collection, etc.
  • SCF stem cell growth factor
  • NEF nerve growth factor
  • IL-6 interleukin-6
  • IL-7 interleukin-7
  • TNF tumor necrosis factor
  • IFN interferon
  • the object of the present invention is to provide a kind of cell protein extract with cell repair effect, and its preparation comprises the steps:
  • the solvent is selected from physiological saline, 5% glucose solution, phosphate buffer saline Any one of (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof;
  • step (3) After separating the cell lysate prepared in step (2), the obtained separation liquid is filtered through 0.45um and 0.22um filter membranes successively to obtain the product.
  • the culture medium of step (1) contains DMEM/F1242-45%, RPMI 1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5 -10ug/mL, fibroblast growth factor (FGF) 5-10ug/mL, insulin transferrin 5-10ug/mL, compound amino acid (18AA) 0.02-0.05% and 3-8 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • the culture medium of step (1) contains DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug/mL, fibroblast Cell growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05%, and 4-6 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • the mesenchymal subcultured cell density in step (1) is 2.0 ⁇ 10 6 -2.0 ⁇ 10 7 cells/mL, preferably 5.0 ⁇ 10 6 -1.0 ⁇ 10 7 cells/mL.
  • the passaged mesenchymal cells in step (1) are cultured in the culture medium for 30min-13h, preferably 45min-12h.
  • the solvent for washing cells in step (1) is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer Or a combination thereof, the number of times of washing the cells is 2-5 times, preferably 3-4 times.
  • the separation described in step (1) is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation condition is 1000-2000rpm*3-15min, Preferably it is 1200rpm-1500rpm*5-10min.
  • the ultrasonic conditions in step (2) are: working at 2°C-8°C, 25kHZ, 360W for 3s, followed by a gap of 1s, ultrasonic treatment for 1-5min.
  • the separation in step (3) is selected from any one of 2000-8000rpm*10-30min centrifugation, multistage centrifugation, multistage filtration or a combination thereof, preferably 3000-7000rpm*15-25min .
  • the multistage centrifugation in step (3) is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min in sequence.
  • the filter membrane pore size of the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.
  • the protein extract prepared in step (3) is frozen, preferably at -40°C to -20°C.
  • a lyoprotectant is added to the filtrate collected in step (3), and lyophilized to obtain the product, wherein the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerol , Sucrose, Trehalose, Glucose, Lactose, Maltose, Dextran, Tricaprylic Glyceride (HES), Polyethylene Glycol, Ethylene Diene, Phosphate, Acetate, Citrate, Sorbitol, Starch any one or combination thereof.
  • the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerol , Sucrose, Trehalose, Glucose, Lactose, Maltose, Dextran, Tricaprylic Glyceride (HES), Polyethylene Glycol, Ethylene Diene, Phosphate, Acetate, Citrate, Sorbitol, Starch any one or combination
  • the lyophilized preparation contains 0.7-7%, preferably 1-5%, of the lyoprotectant in terms of mass percentage.
  • a protein stabilizer is optionally added to the filtrate collected in step (3), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.
  • the pH of the freeze-dried preparation is 6-8, preferably pH 7-7.5.
  • the freeze-dried preparation is reconstituted with an isotonic solution before use, it is smeared, needle-rolled, micro-needled, massaged, intravenously injected, intramuscularly injected, subcutaneously injected, acupoint injected, or lumbar puncture.
  • the isotonic solution is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof.
  • the culture of the passaged mesenchymal stem cells or the culture of the primary mesenchymal stem cells adopts a culture method in the art.
  • the cultivation of the passaged mesenchymal stem cells includes the following steps: adding primary mesenchymal stem cells to the passage medium at an initial density of 5.0 ⁇ 10 5 -5.0 ⁇ 10 6 cells/ml culture medium at 37.0°C ⁇ 0.5°C and 5% ⁇ 1.0% CO 2 for 10-15 days, and after every 2-3 days, half of the subculture medium was replaced after the subculture medium was observed to turn yellow.
  • the subculture medium contains 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin in DMEM/F12 medium.
  • the cultivation of the primary mesenchymal stem cells comprises the following steps:
  • the tissue is dissected, and the Huatong glue layer tissue is taken, cut into small pieces of 3 mm 3 , centrifuged, cleaned, and the tissue pieces are collected, and placed in 10% fetal bovine serum FBS, 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 , half of the medium was replaced every 2-3 days, Culture until the tissue block climbs out of the cells;
  • 10% fetal bovine serum FBS 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium
  • the object of the present invention is to provide a kind of preparation method of the cell protein extract with cell repair effect, comprises the steps:
  • the solvent is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, Tris buffer or a combination thereof;
  • step (3) After separating the cell lysate prepared in step (2), the obtained separation liquid is filtered through 0.45um and 0.22um filter membranes successively to obtain the product.
  • the culture medium of step (1) contains DMEM/F1242-45%, RPMI 1640 42-45%, bovine serum albumin (BSA) 0.5-1.5%, epidermal growth factor (EGF) 5 -10ug/mL, fibroblast growth factor (FGF) 5-10ug/mL, insulin transferrin 5-10ug/mL, compound amino acid (18AA) 0.02-0.05% and 3-8 ⁇ mol/L of stressors.
  • BSA bovine serum albumin
  • the culture medium of step (1) contains DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug/mL, fibroblast Growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05%, and 4-6 ⁇ mol/L stressors.
  • BSA bovine serum albumin
  • EGF epidermal growth factor
  • FGF fibroblast Growth factor
  • insulin transferrin 10ug/mL
  • compound amino acid (18AA) 0.05% compound amino acid (18AA) 0.05%
  • the mesenchymal subcultured cell density in step (1) is 2.0 ⁇ 10 6 -2.0 ⁇ 10 7 cells/mL, preferably 5.0 ⁇ 10 6 -1.0 ⁇ 10 7 cells/mL.
  • the passaged mesenchymal cells in step (1) are cultured in the culture medium for 30min-13h, preferably 45min-12h.
  • the solvent for washing cells in step (1) is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer Or a combination thereof, the number of times of washing the cells is 2-5 times, preferably 3-4 times.
  • the separation described in step (1) is selected from any one of centrifugation and filtration or a combination thereof, wherein the centrifugation condition is 1000-2000rpm*3-15min, preferably 1200rpm-1500rpm* 5-10min.
  • the culture of the passaged mesenchymal stem cells or the culture of the primary mesenchymal stem cells adopts a culture method in the art.
  • the cultivation of the passaged mesenchymal stem cells includes the following steps: adding primary mesenchymal stem cells to the passage medium at an initial density of 5.0 ⁇ 10 5 -5.0 ⁇ 10 6 cells/ml , and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 for 10-15 days, every 2-3 days, after observing the yellowing of the subculture medium, half the amount was replaced The subculture medium was changed, wherein the subculture medium contained DMEM/F12 medium containing 10% FBS, 100 U/ml penicillin and 100 ug/ml streptomycin.
  • the cultivation of the primary mesenchymal stem cells comprises the following steps:
  • the tissue is dissected, and the Huatong glue layer tissue is taken, cut into small pieces of 3 mm 3 , centrifuged, cleaned, and the tissue pieces are collected, and placed in 10% fetal bovine serum FBS, 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium, and then cultured at 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0% CO 2 , half of the medium was replaced every 2-3 days, Culture until the tissue block climbs out of the cells;
  • 10% fetal bovine serum FBS 100ug/ ml penicillin, 100ug/ml streptomycin in DMEM/F12 medium
  • the ultrasonic conditions in step (2) are: working at 2°C-8°C, 25kHZ, 360W for 3s, followed by a gap of 1s, ultrasonic treatment for 1-5min.
  • the separation in step (3) is selected from any one of 2000-8000rpm*10-30min centrifugation, multistage centrifugation, multistage filtration or a combination thereof, preferably 3000-7000rpm*15-25min .
  • the multistage centrifugation in step (3) is 3000-4000rpm*3-5min, 5000-6000rpm*3-5min and 7000rpm*5-8min in sequence.
  • the filter membrane pore size of the multi-stage filtration is selected from any one of 80um, 50um, 30um, 10um, and 5um.
  • the protein extract prepared in step (3) is frozen, Preferably frozen at -40°C to -20°C.
  • a lyoprotectant is added to the filtrate collected in step (3), and lyophilized to obtain the product, wherein the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerol , Sucrose, Trehalose, Glucose, Lactose, Maltose, Dextran, Tricaprylic Glyceride (HES), Polyethylene Glycol, Ethylene Diene, Phosphate, Acetate, Citrate, Sorbitol, Starch any one or combination thereof.
  • the lyoprotectant is selected from mannitol, sorbitol, dextran, glycerol , Sucrose, Trehalose, Glucose, Lactose, Maltose, Dextran, Tricaprylic Glyceride (HES), Polyethylene Glycol, Ethylene Diene, Phosphate, Acetate, Citrate, Sorbitol, Starch any one or combination
  • the lyophilized preparation contains 0.7-7%, preferably 1-5%, of the lyoprotectant in terms of mass percentage.
  • a protein stabilizer is optionally added to the filtrate collected in step (3), wherein the protein stabilizer is selected from any one of albumin, zinc salt, and aluminum salt.
  • the pH of the freeze-dried preparation is 6-8, preferably pH 7-7.5.
  • the freeze-dried preparation is reconstituted with an isotonic solution before use, it is smeared, needle-rolled, micro-needled, massaged, intravenously injected, intramuscularly injected, subcutaneously injected, acupoint injected, or lumbar puncture.
  • the isotonic solution is selected from any one of physiological saline, 5% glucose solution, phosphate buffer saline (PBS), TBPS buffer, TBST buffer, and Tris buffer or a combination thereof.
  • Another object of the present invention is to provide a cell repair composition, which is composed of the cell protein extract with cell repair function of the present invention and a pharmaceutically acceptable carrier.
  • the amount or type of the pharmaceutically acceptable carrier of the present invention depends on the physical and chemical properties and content of the active ingredients in the composition, the type of preparation, the dissolution and bioavailability of the preparation and other factors.
  • composition of the present invention can be in various dosage forms in the art, and can be prepared by using the formulation technology in the art.
  • the composition is selected from any one of lyophilized agent, gel, nasal spray, ointment, cream, emulsion, liquid dressing, injection and suppository.
  • the administration method of the composition is selected from any one or a combination of smearing, needle rolling, microneedle, massage, intravenous injection, intramuscular injection, subcutaneous injection, acupoint injection, lumbar puncture.
  • Another object of the present invention is to provide the cell protein extract with cell repair effect or its composition of the present invention for preparing cell repair, joint repair, cartilage repair, skin damage cell repair, nerve damage cell repair, organ damage Repairing, repairing lung fibrosis, repairing liver damage, repairing kidney damage, repairing ovary, anti-Crohn's disease, anti-sub-health, anti-aging any one of the medicines or products.
  • Another object of the present invention is to provide the application of compound 1-16 for stress-induced stem cells to produce functional proteins with repair function.
  • the repair is any one of cell repair, hair follicle repair, joint repair, and nerve repair.
  • the present invention when the present invention relates to the percentage between liquids and liquids, said percentages are volume/volume percentages; when the present invention relates to the percentages between liquids and solids, Said percentages are volume/weight percentages; when the present invention relates to percentages between solids and liquids, said percentages are weight/volume percentages; the rest are weight/weight percentages.
  • the present invention adopts following method to detect:
  • the present invention has the following beneficial effects:
  • the present invention scientifically screens the culture medium containing stressors to induce mesenchymal stem cells to produce cell proteins with cell repair effects.
  • the obtained cell protein extracts have the functions of repairing nerve damage, repairing joint damage, repairing damaged hair follicle cells, Repair skin damage, repair pulmonary fibrosis, repair liver damage, repair kidney damage, repair sub-health, repair premature ovarian failure, prevent and treat Crohn's disease, anti-aging, etc., and have high purity, good stability, safe and effective, and effectively solve living Cells need to be refrigerated and their activity is limited by the time of cell viability, among other advantages.
  • the preparation method of the present invention has the advantages of simple operation, environmental protection, better cost, and suitability for industrial production.
  • Fig. 1 cell protein extract of the present invention is to the research of repairing action of oxidative damage skin
  • Fig. 2 cell protein extract of the present invention is to the repair effect research of skin damage caused by UVB irradiation
  • FIG. 3 Effect of cell protein extract of the present invention on oxide (IOD) generation in skin damage caused by UVB irradiation.
  • Mean ⁇ SD (n 3). ## indicates p ⁇ 0.01 compared with blank control (BC) group; *p ⁇ 0.05 compared with NC (negative control) group, ** indicates p ⁇ 0.01 compared with NC (negative control) group;
  • Fig. 6 protein extract of the present invention is to the repair effect research of medial condyle joint damage
  • the cultivation of primary mesenchymal stem cells includes the following steps:
  • Subculture of primary mesenchymal stem cells Add primary mesenchymal stem cells to a medium containing 10% FBS, 100U/ml penicillin and 100ug/ml streptomycin at an initial density of 1.0 ⁇ 10 5 -6.0 ⁇ 10 5 cells/ml In DMEM/F12 culture medium, put it in the condition of 37.0°C ⁇ 0.5°C, 5% ⁇ 1.0%CO 2 and culture it for 10-15 days, every 2-3 days, observe that the medium turns yellow, half volume Replace medium.
  • the preparation of compound 1-16 refers to literature 1 (New limonophyllines A-C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines, Arch.Pharm.Res.(2018)41:431-437).
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug at a density of 5.0 ⁇ 10 6 cells/mL /mL, fibroblast growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05% and 5 ⁇ mol/L in the culture medium of compound 16, then place it in 37 °C, After culturing for 30 minutes under the condition of 5% CO 2 , centrifuge at 1200 rpm for 5 minutes, wash with PBS for 3 times, and collect the cells;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 8.0 ⁇ 10 6 cells/mL, operate ultrasonically for 3 seconds with a gap of 1 second at 2-8°C, 25kHz, and 360W, and sonicate for 2 minutes to obtain Cell Lysates;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • Embodiment 2 Preparation of cell protein extract freeze-dried preparation of the present invention
  • Embodiment 3 The preparation of cell protein extract of the present invention
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug at a density of 8.0 ⁇ 10 6 cells/mL /mL, fibroblast growth factor (FGF) 10ug/mL, pancreas
  • BSA bovine serum albumin
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • pancreas In the culture medium of insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05% and 5 ⁇ mol/L compound 16, it was placed at 37°C and 5% CO 2 After cultivating for 4h, it was placed in Centrifuge at 1200rpm*5min, wash 3 times with PBS, and collect the cells;
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 1.0 ⁇ 10 7 cells/mL, sonicate for 3s with a gap of 1s at 2-8°C, 25kHz, 360W, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • Embodiment 4 Preparation of cell protein extract freeze-dried preparation of the present invention
  • Embodiment 5 The preparation of cell protein extract of the present invention
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug at a density of 5.0 ⁇ 10 6 cells/mL /mL, fibroblast growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05% and 5 ⁇ mol/L in the culture medium of compound 16, then place it in 37 °C, After culturing for 12 hours under the condition of 5% CO 2 , centrifuge at 1200 rpm for 5 minutes, wash with PBS for 3 times, and collect the cells;
  • BSA bovine serum albumin
  • step (1) Disperse the cells collected in step (1) at a density of 1.0 ⁇ 10 7 cells/mL in In normal saline, under the conditions of 2-8°C, 25kHz, 360W, sonicate for 3s, with an interval of 1s, and sonicate for 2min, to prepare cell lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • Embodiment 6 The preparation of cell protein extract freeze-dried preparation of the present invention
  • Embodiment 7 The preparation of cell protein extract of the present invention
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, epidermal growth factor (EGF) 10ug at a density of 5.0 ⁇ 10 6 cells/mL /mL, fibroblast growth factor (FGF) 10ug/mL, insulin-transferrin 10ug/mL, compound amino acid (18AA) 0.05% and 5 ⁇ mol/L in the medium of compound 13, then place it in 37 °C, After culturing for 30 minutes under the condition of 5% CO 2 , centrifuge at 1200 rpm for 5 minutes, wash with PBS for 3 times, and collect the cells;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 7.0 ⁇ 10 6 cells/mL, and ultrasonically work for 3 seconds with an interval of 1 second at 2-8°C, 25 kHz, and 360 W for 2 minutes to obtain Cell Lysates;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product. according to need If necessary, the resulting cellular protein extract is lyophilized.
  • Sorbitol was added to the cell protein extract prepared in Example 7, stirred, mixed evenly, and then freeze-dried.
  • the resulting freeze-dried preparation contained 3% sorbitol (m/m).
  • Embodiment 9 Preparation of cell protein extract of the present invention
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, and epidermal growth factor (EGF) 10ug at a density of 1.0 ⁇ 107 /mL /mL, fibroblast growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05% and the medium of compound 14 of 8 ⁇ mol/L, then it is placed in 37 °C, After culturing for 4 hours under the condition of 5% CO 2 , it was centrifuged at 1200 rpm for 5 minutes, washed with PBS for 3 times, and the cells were collected;
  • BSA bovine serum albumin
  • EGF epidermal growth factor
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 5.0 ⁇ 107 cells/mL, sonicate at 2-8°C, 25kHz, 360W for 3s, with an interval of 1s, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • the obtained freeze-dried preparation contains 1% dextran (m/m).
  • Embodiment 11 Preparation of cell protein extract of the present invention
  • the preparation method of cell protein extract of the present invention comprises the steps:
  • the passaged mesenchymal cells were added to a medium containing DMEM/F12 45%, RPMI 1640 45%, bovine serum albumin (BSA) 0.5%, and epidermal growth factor (EGF) 10ug at a density of 1.0 ⁇ 107 /mL /mL, fibroblast growth factor (FGF) 10ug/mL, insulin transferrin 10ug/mL, compound amino acid (18AA) 0.05% and the medium of compound 15 of 8 ⁇ mol/L, then it is placed in 37 °C, After culturing for 12 hours under the condition of 5% CO 2 , centrifuge at 1200 rpm for 5 minutes, wash with PBS for 3 times, and collect the cells;
  • BSA bovine serum albumin
  • step (2) Disperse the cells collected in step (1) in physiological saline at a density of 5.0 ⁇ 107 cells/mL, sonicate at 2-8°C, 25kHz, 360W for 3s, with an interval of 1s, and sonicate for 2min to prepare the cells lysate;
  • step (3) Centrifuge the cell lysate prepared in step (2) at 7000 rpm for 20 min, and filter the obtained centrifugate through 0.45um and 0.22um membrane filters in turn to obtain the product.
  • Test Example 1 Research on the repairing effect of cell protein extract of the present invention on oxidative damage skin
  • 3D skin model Commercially available from EpiKutis.
  • SLS working solution Weigh 0.0080g SLS, dissolve it in 2mL PBS solution, filter through 0.22 ⁇ m, and prepare 0.4% SLS mother solution. Draw 0.5mL 0.4% SLS mother solution, add 0.5mL PBS, and make it into 0.2% SLS working solution.
  • WY14643 working solution Weigh 10mg WY14643 (PPAR ⁇ agonist), dissolve it in 1mL DMSO, and prepare 30mM WY14643 mother solution. Then 10 ⁇ L of WY14643 mother solution (30 mM) was added to 6 mL of model culture solution to prepare 50 ⁇ M WY14643 working solution.
  • Model culture medium DMEM basal medium.
  • Blank control (BC) without any treatment, the skin model was placed in a CO 2 incubator (37°C, 5% CO 2 ) and incubated for 48 hours;
  • Negative control Add 25 ⁇ L of 0.2% SLS working solution on the surface of the skin model, and place it in a CO 2 incubator (37°C, 5% CO 2 ) for 24 hours;
  • PC Positive control
  • Test group Add 25 ⁇ L of 0.2% SLS working solution on the surface of the skin model, place it in a CO 2 incubator (37°C, 5% CO 2 ) and incubate for 24 hours; add 25 ⁇ L of 1% cell protein extract solution (with The cell protein extract freeze-dried powder of Example 2 was made into a 1% solution with physiological saline), and then placed in a CO 2 incubator (37° C., 5% CO 2 ) for incubation for 24 hours.
  • the protein extract of the present invention has obvious repairing effect on oxidative damage skin.
  • 3D skin model Commercially available from EpiKutis.
  • Model culture medium DMEM basal medium.
  • VE working solution draw 0.5g VE stock solution, dissolve it in 10mL absolute ethanol, and prepare 5% mother solution; absorb 100 ⁇ L 5% mother solution, dissolve it in 10mL model culture medium, and prepare 0.05% VE working solution liquid.
  • Blank control (BC) without any treatment, the skin model was placed in a CO 2 incubator (37°C, 5% CO 2 ) and incubated for 48 hours;
  • Negative control After irradiating the surface of the skin model with UVB at a dose of 600mJ/cm 2 , place it in a CO 2 incubator (37°C, 5% CO 2 ) and incubate it for 24h; add 25 ⁇ L of 0.2% SLS to work solution, and incubated in a CO 2 incubator (37°C, 5% CO 2 ) for 24 hours;
  • PC Positive control
  • Test group After the skin model surface was irradiated with UVB at a dose of 600mJ/cm 2 , it was placed in a CO 2 incubator (37°C, 5% CO 2 ) and incubated for 24 hours; 25ul of 1% cell protein extract solution was added (The lyophilized powder of cell protein extract prepared in Example 4 of the present invention was made into a 1% solution with physiological saline), and then placed in a CO 2 incubator (37° C., 5% CO 2 ) and incubated for 24 hours.
  • Test Example 3 Research on the repairing effect of the cell protein extract of the present invention on joint damage and cartilage damage
  • a 12-month-old male goat with a normal feeding weight of 47 kg was given general anesthesia, and a median incision of 8 cm was made on the left medial condyle, and the medial edge of the patellar tendon was separated to the joint, exposing the medial femoral condyle, the medial tibial plateau, and the surface of the articular cartilage.
  • Smooth use a 3mm ball drill to make a 0.3 ⁇ 0.3 ⁇ 0.6cm (length ⁇ width ⁇ depth) cartilage injury wound on the medial femoral condyle, and suture the incision (see Figure 6).
  • test sheep were killed under anesthesia, and the cartilage slices were taken (see Figure 6-7).
  • the cell protein extract of the invention has an excellent effect of repairing joint damage, and is safe and effective.

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Abstract

本发明涉及一种具有细胞修复功效的细胞蛋白提取物，其制备包括如下步骤：(1)将密度为1.0×106个/mL-5.0×107个/mL的间充质传代细胞置于含有DMEM/F12 40-50％、RPMI 1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10min-14h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合；(2)将收集细胞置于2℃-8℃条件下超声处理，制得细胞裂解液，过滤，即得。本发明所得的细胞蛋白提取物或其组合物可促进组织内源性修复，可用于修复神经损伤、修复关节损伤、修复受损毛囊细胞、修复皮肤损伤，并具有纯度高、稳定性好、安全有效、有效解决活细胞需要冷藏且其活性受细胞活力时间限制等优点。

Description

一种细胞修复蛋白提取物及其制备方法和其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种细胞修复蛋白提取物及其制备方法和其应用。
背景技术
细胞是生命活动的基本单位和机体健康的基础。当机体的氧化还原平衡遭到破坏时，会引起氧化还原信号和控制的中断，引发氧化应激损伤而引发多种疾病。及时修复损伤细胞可以显著改善、治疗相关病变。
细胞及微生物受到外界刺激及外源应激物(包括冷、热、酸、碱、电流、辐射、化学物质等)应激诱导时会因应激反应而诱导细胞及微生物产生应激蛋白。文献1(New limonophyllines A-C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2cell lines，Arch.Pharm.Res.(2018)41:431–437)公开了从芸香科植物(Atalantia monophylla)提取制得的化合物1-16具有抑制肿瘤细胞生长等活性。
间充质干细胞(mesenchymal stem cells，MSCs)具有自我复制和多向分化潜能，广泛存在于骨髓、脂肪、滑膜、牙髓、羊水、胎盘、脐带、胚胎、脐带血、羊膜、外周血、肌肉、尿液等组织，具有来源广泛、无需配型、感染率低、分化潜能强、增殖能力强、采集方便等 特点，可产生干细胞生长因子(SCF)、神经生长因子(NGF)、白介素-6(IL-6)、白介素-7(IL-7)、肿瘤坏死因子(TNF)、干扰素(IFN)等活性因子，参与调节细胞生长、细胞凋亡、细胞分化、抗病毒、免疫成熟等过程，可用于免疫调节、组织修复及治疗急性肺损伤、重症肺炎、急性呼吸窘迫综合征等疾病。但MSCs产品需在其生产、储运和应用等环节采用冷藏方式，且其细胞活力保持≤12h，而限制其治疗应用。为此，需要开发安全有效的皮肤修复药物以满足临床需求。
发明内容
本发明的目的在于提供一种具有细胞修复功效的细胞蛋白提取物，其制备包括如下步骤：
(1)将密度为1.0×106个/mL-5.0×107个/mL的间充质传代细胞置于含有DMEM/F12 40-50％、RPMI 1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10min-14h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合，

(2)将收集细胞按照密度为5.0×106个/mL-1.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液 (PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
(3)将步骤(2)制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
本发明的优选技术方案中，步骤(1)的培养基中含有DMEM/F1242-45％、RPMI 1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF)5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和3-8μmol/L的应激物。
本发明的优选技术方案中，步骤(1)的培养基中含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL，胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和4-6μmol/L的应激物。
本发明的优选技术方案中，步骤(1)的间充质传代细胞密度为2.0×106-2.0×107个/mL，优选为5.0×106-1.0×107个/mL。
本发明的优选技术方案中，步骤(1)的间充质传代细胞在培养基中培养30min-13h，优选为45min-12h。
本发明的优选技术方案中，步骤(1)中洗涤细胞的溶剂选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合,洗涤细胞次数为2-5次，优选为3-4次。
本发明的优选技术方案中，步骤(1)所述的分离选自离心、过滤的任一种或其组合，其中，所述离心条件为1000-2000rpm*3-15min， 优选为1200rpm-1500rpm*5-10min。
本发明的优选技术方案中，步骤(2)的超声条件为：在2℃-8℃、25kHZ、360W条件下工作3s再间隙1s，超声处理1-5min。
本发明的优选技术方案中，步骤(3)所述分离选自2000-8000rpm*10-30min离心、多级离心、多级过滤的任一种或其组合，优选为3000-7000rpm*15-25min。
本发明的优选技术方案中，步骤(3)的多级离心依次为3000-4000rpm*3-5min、5000-6000rpm*3-5min和7000rpm*5-8min。
本发明的优选技术方案中，所述多级过滤的滤膜孔径选自80um、50um、30um、10um、5um的任一种。
本发明的优选技术方案中，将步骤(3)制得的蛋白提取物冻存，优选冻存于-40℃至-20℃。
本发明的优选技术方案中，在步骤(3)收集到的滤液中加入冻干保护剂，冻干，即得，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、甘油、蔗糖、海藻糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯(HES)、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
本发明的优选技术方案中，以质量百分比计，所述冻干制剂中含有冻干保护剂0.7-7％，优选为1-5％。
本发明的优选技术方案中，将步骤(3)收集的滤液中任选地加入蛋白稳定剂，其中，所述蛋白稳定剂选自白蛋白、锌盐、铝盐的任一种。
本发明的优选技术方案，所述冻干制剂pH6-8，优选为pH7-7.5。
本发明的优选技术方案中，所述冻干制剂临用前用等渗溶液复溶后，采用涂抹、滚针、微针、按摩、静脉注射、肌肉注射、皮下注射、穴位注射、腰椎穿刺的任一种或其组合方式使用，其中，所述的等渗溶液选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合。
本发明的优选技术方案中，所述间充质传代干细胞的培养或原代间充质干细胞的培养采用本领域的培养方法。
本发明的优选技术方案中，所述间充质传代干细胞的培养包括下述步骤：将原代间充质干细胞按照初始密度为5.0×105-5.0×106个/ml加入到传代培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每隔2-3天，观察传代培养基变黄后，半量更换传代培养基，其中，所述传代培养基含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基。
本发明的优选技术方案中，所述原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于含10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养，每间隔2-3天半量更换培养基，培养至组织块爬出细胞；
2)振摇，收集低层细胞用PBS清洗后，加入0.25％的胰蛋白酶 消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打，1200-1500rpm/min*5-8min离心后，收集细胞，即得。
本发明的目的在于提供一种具有细胞修复功效的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将密度为1.0×106个/mL-5.0×107个/mL的间充质传代细胞置于含有DMEM/F12 40-50％、RPMI 1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10min-14h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合；
(2)将收集细胞按照密度为5.0×106个/mL-1.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
(3)将步骤(2)制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
本发明的优选技术方案中，步骤(1)的培养基中含有DMEM/F1242-45％、RPMI 1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF)5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和 3-8μmol/L的应激物。
本发明的优选技术方案中，步骤(1)的培养基中含有DMEM/F1245％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL，胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和4-6μmol/L的应激物。
本发明的优选技术方案中，步骤(1)的间充质传代细胞密度为2.0×106-2.0×107个/mL，优选为5.0×106-1.0×107个/mL。
本发明的优选技术方案中，步骤(1)的间充质传代细胞在培养基中培养30min-13h，优选为45min-12h。
本发明的优选技术方案中，步骤(1)中洗涤细胞的溶剂选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合,洗涤细胞次数为2-5次，优选为3-4次。
本发明的优选技术方案中，步骤(1)所述的分离选自离心、过滤的任一种或其组合，其中，所述离心条件为1000-2000rpm*3-15min，优选为1200rpm-1500rpm*5-10min。
本发明的优选技术方案中，所述间充质传代干细胞的培养或原代间充质干细胞的培养采用本领域的培养方法。
本发明的优选技术方案中，所述间充质传代干细胞的培养包括下述步骤：将原代间充质干细胞按照初始密度为5.0×105-5.0×106个/ml加入到传代培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每隔2-3天，观察传代培养基变黄后，半量更 换传代培养基，其中，所述传代培养基含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基。
本发明的优选技术方案中，所述原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于含10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养，每间隔2-3天半量更换培养基，培养至组织块爬出细胞；
2)振摇，收集低层细胞用PBS清洗后，加入0.25％的胰蛋白酶消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打，1200-1500rpm/min*5-8min离心后，收集细胞，即得。
本发明的优选技术方案中，步骤(2)的超声条件为：在2℃-8℃、25kHZ、360W条件下工作3s再间隙1s，超声处理1-5min。
本发明的优选技术方案中，步骤(3)所述分离选自2000-8000rpm*10-30min离心、多级离心、多级过滤的任一种或其组合，优选为3000-7000rpm*15-25min。
本发明的优选技术方案中，步骤(3)的多级离心依次为3000-4000rpm*3-5min、5000-6000rpm*3-5min和7000rpm*5-8min。
本发明的优选技术方案中，所述多级过滤的滤膜孔径选自80um、50um、30um、10um、5um的任一种。
本发明的优选技术方案中，将步骤(3)制得的蛋白提取物冻存， 优选冻存于-40℃至-20℃。
本发明的优选技术方案中，在步骤(3)收集到的滤液中加入冻干保护剂，冻干，即得，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、甘油、蔗糖、海藻糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯(HES)、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
本发明的优选技术方案中，以质量百分比计，所述冻干制剂中含有冻干保护剂0.7-7％，优选为1-5％。
本发明的优选技术方案中，将步骤(3)收集的滤液中任选地加入蛋白稳定剂，其中，所述蛋白稳定剂选自白蛋白、锌盐、铝盐的任一种。
本发明的优选技术方案，所述冻干制剂pH6-8，优选为pH7-7.5。
本发明的优选技术方案中，所述冻干制剂临用前用等渗溶液复溶后，采用涂抹、滚针、微针、按摩、静脉注射、肌肉注射、皮下注射、穴位注射、腰椎穿刺的任一种或其组合方式使用，其中，所述的等渗溶液选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合。
本发明的另一目的在于提供一种细胞修复组合物，所述组合物由本发明的具细胞修复功效的细胞蛋白提取物和药学上可接受的载体组成。
本发明的药学上可接受载体的用量或其种类根据组合物中有效成分的理化性质和含量、制剂类型、制剂的溶出及生物利用度等因素而定。
本发明的组合物可为本领域的各种剂型，并可采用本领域的制剂技术制备得到。
本发明的优选技术方案中，所述组合物选自冻干剂、凝胶剂、鼻喷剂、膏剂、霜剂、乳剂、液体敷料、注射剂、栓剂的任一种。
本发明的优选技术方案中，所述组合物的给药方式选自涂抹、滚针、微针、按摩、静脉注射、肌肉注射、皮下注射、穴位注射、腰椎穿刺的任一种或其组合。
本发明的另一目的在于提供本发明所述具有细胞修复功效的细胞蛋白提取物或其组合物用于制备细胞修复、关节修复、软骨修复、皮肤损伤细胞修复、神经损伤细胞修复、脏器损伤修复、肺纤维化修复、肝损伤修复、肾损伤修复、卵巢修复、抗克罗恩病、抗亚健康、抗衰的任一种的药物或制品中的应用。
本发明的另一目的在于提供化合物1-16用于应激诱导干细胞产生具有修复功效的功能蛋白的应用。
本发明的优选技术方案中，所述修复为细胞修复、毛囊修复、关节修复、神经修复中的任一种。
除非另有说明，本发明涉及液体与液体之间的百分比时，所述的百分比为体积/体积百分比；本发明涉及液体与固体之间的百分比时， 所述百分比为体积/重量百分比；本发明涉及固体与液体之间的百分比时，所述百分比为重量/体积百分比；其余为重量/重量百分比。
除非另有说明，本发明采用如下方法进行检测：
1.间充质干细胞MSCs鉴定：《Standards for the culture and quality control of umbilical cord mesenchymal stromal cells for neurorestorative clinical application》
2.采用商购试剂盒检测活性氧(IOD)、超氧化物歧化酶SOD、丙二醛MDA水平。
与现有技术相比，本发明具有下述有益效果：
1、本发明科学筛选含有应激物的培养基应激诱导间充质干细胞产生具有细胞修复功效的细胞蛋白，所得的细胞蛋白提取物具有修复神经损伤、修复关节损伤、修复受损毛囊细胞、修复皮肤损伤、修复肺纤维化、修复肝损伤、修复肾损伤、修复亚健康、修复卵巢早衰、防治克罗恩病、抗衰等，并具有纯度高、稳定性好、安全有效、有效解决活细胞需要冷藏且其活性受细胞活力时间限制等优点。
2、本发明的制备方法具有操作简便，绿色环保，成本更优，适用于工业化生产等优点。
附图说明
图1本发明的细胞蛋白提取物对氧化损伤皮肤的修复作用研究；
图2本发明的细胞蛋白提取物对UVB照射所致皮肤损伤的修复作用研究；
图3本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中氧化物(IOD)生成的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图4本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中超氧化物歧化酶SOD生成的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图5本发明的细胞蛋白提取物对UVB照射所致皮肤损伤中MDA水平的影响。平均值±SD(n＝3)。##表示与空白对照(BC)组相比p<0.01；*与NC(阴性对照)组相比p<0.05，**表示与NC(阴性对照)组相比，p<0.01；
图6本发明的蛋白提取物对内髁关节损伤的修复作用研究；
图7本发明的蛋白提取物对平台关节损伤的修复作用研究。
具体实施方式
下面结合具体实施例对本发明的详细内容做进一步解释和描述，但并不以此限制本发明的保护范围。
1、原代间充质干细胞的培养
原代间充质干细胞的培养包括下述步骤：
1)将脐带清洗消毒后，组织解剖，取华通胶层组织，将其切成3mm3的小块，离心，清洗，收集组织块，将其置于培养瓶中，加入含 10％胎牛血清FBS、100ug/ml青霉素、100ug/ml链霉素的DMEM/F12培养基，再将其置于37℃、5％CO2条件下培养，促进其贴壁，每间隔2-3天,观察培养基变黄后，半量更换培养基，培养10-12天，至组织块边上可见细胞爬出；
2)轻轻摇晃，使组织块掉落，分别收集组织块和低层细胞，其中，将收集的组织块再贴壁培养；
3)将收集的低层细胞用PBS清洗后，加入适量0.25％胰蛋白酶消化2min-3min，加入等体积的胰蛋白酶终止液停止消化，吸管轻轻吹打瓶底，1500rpm/min*5min离心后，收集细胞，即得。
2、原代间充质干细胞的传代培养
原代间充质干细胞的传代培养：将原代间充质干细胞按照初始密度为1.0×105-6.0×105个/ml加入到含有10％FBS、100U/ml青霉素和100ug/ml链霉素的DMEM/F12培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10-15天，每间隔2-3天，观察培养基变黄后，半量更换培养基。
3、化合物1-16的制备参照文献1(New limonophyllines A-C from the stem of Atalantia monophylla and cytotoxicity against cholangiocarcinoma and HepG2 cell lines，Arch.Pharm.Res.(2018)41:431–437)。
实施例1本发明细胞蛋白提取物的制备
本发明细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为5.0×106个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物16的培养基中，再将其置于37℃、5％CO2条件下培养30min后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为8.0×106个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声工作3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例2本发明细胞蛋白提取物冻干制剂的制备
在实施例1制得的细胞蛋白提取物中加入所需量的甘露醇，搅拌，混合均匀后，冻干，所得冻干制剂中含2％的甘露醇(m/m)。
实施例3本发明细胞蛋白提取物的制备
本发明的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为8.0×106个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰 岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物16的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为1.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例4本发明细胞蛋白提取物冻干制剂的制备
在实施例3制得的细胞蛋白提取物中加入所需量的甘露醇，搅拌，混合均匀后，冻干，所得冻干制剂中含5％的甘露醇(m/m)。
实施例5本发明细胞蛋白提取物的制备
本发明细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为5.0×106个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物16的培养基中，再将其置于37℃、5％CO2条件下培养12h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为1.0×107个/mL分散于 生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例6本发明细胞蛋白提取物冻干制剂的制备
在实施例5制得的细胞蛋白提取物中加入所需量的甘露醇，搅拌，混合均匀后，冻干，所得冻干制剂中含2％的甘露醇(m/m)。
实施例7本发明细胞蛋白提取物的制备
本发明的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为5.0×106个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和5μmol/L的化合物13的培养基中，再将其置于37℃、5％CO2条件下培养30min后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为7.0×106个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声工作3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。可根据需 要，将制得的细胞蛋白提取物冻干。
实施例8本发明细胞蛋白提取物冻干制剂的制备
在实施例7制得的细胞蛋白提取物中加入山梨醇，搅拌，混合均匀后，冻干，所得冻干制剂中含有3％的山梨醇(m/m)。
实施例9本发明细胞蛋白提取物的制备
本发明的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为1.0×107个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和8μmol/L的化合物14的培养基中，再将其置于37℃、5％CO2条件下培养4h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为5.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例10本发明细胞蛋白提取物冻干制剂的制备
在实施例9制得的细胞蛋白提取物中加入右旋糖酐，搅拌，混合 均匀后，冻干，所得冻干制剂中含有1％的右旋糖酐(m/m)。
实施例11本发明细胞蛋白提取物的制备
本发明的细胞蛋白提取物的制备方法，包括如下步骤：
(1)将间充质传代细胞按照密度为1.0×107个/mL加入到含有DMEM/F12 45％、RPMI 1640 45％、牛血清蛋白(BSA)0.5％、表皮细胞生长因子(EGF)10ug/mL、成纤维细胞生长因子(FGF)10ug/mL、胰岛素转铁蛋白10ug/mL、复方氨基酸(18AA)0.05％和8μmol/L的化合物15的培养基中，再将其置于37℃、5％CO2条件下培养12h后，将其置于1200rpm*5min离心，用PBS洗涤3次后，收集细胞；
(2)将步骤(1)收集的细胞按照密度为5.0×107个/mL分散于生理盐水中，在2-8℃、25kHz、360W条件下超声3s、间隙1s，超声2min，制得细胞裂解液；
(3)将步骤(2)制得的细胞裂解液置于7000rpm*20min离心，将所得的离心液依次经0.45um、0.22um滤膜过滤，即得。
实施例12本发明细胞蛋白提取物冻干制剂的制备
在实施例11制得的细胞蛋白提取物中加入右旋糖酐，搅拌，混合均匀后，冻干，即得，所得冻干制剂中含有2％的右旋糖酐(m/m)。
试验例1本发明细胞蛋白提取物对氧化损伤皮肤的修复作用研究
3D皮肤模型：商购于EpiKutis。
SLS工作液：称取0.0080g SLS，将其溶于2mLPBS溶液中，经0.22μm过滤后，配制成0.4％SLS母液。吸取0.5mL 0.4％SLS母液，加入0.5mL PBS，将其配制成0.2％SLS工作液。
WY14643工作液：称取10mg WY14643(PPARα激动剂)，将其溶于1mLDMSO中，配制成30mM WY14643母液。再将10μL的WY14643母液(30mM)加入到6mL模型培养液中，将其配制成50μM的WY14643工作液。
模型培养液：DMEM基础培养基。
在6孔板中加入0.9mL模型培养液，将3D皮肤模型转移到6孔板中，标注测试编号。
空白对照(BC)：皮肤模型不做任何处理，将其置于CO2培养箱(37℃、5％CO2)中孵育48h后；
阴性对照(NC)：在皮肤模型表面加入25μL 0.2％的SLS工作液，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
阳性对照(PC)：在皮肤模型表面加入25μL 0.2％的SLS工作液，将其置于CO2培养箱(37℃、5％CO2)中孵育24h后；添加25μL 50μM的WY14643工作液后，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
试验组：在皮肤模型表面加入25μL 0.2％的SLS工作液，将其置于CO2培养箱(37℃、5％CO2)中孵育24h后；添加25μL 1％的细胞蛋白提取物溶液(将实施例2的细胞蛋白提取物冻干粉用生理盐水制成1％的溶液)，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h。
孵育48h后，用PBS溶液洗涤并清除皮肤模型内外残留液体。用4％的多聚甲醛固定处理24h后，将模型环切取下，经H&E染色处理后观察。结果见图1。本发明的蛋白提取物对氧化损伤皮肤具有明显的修复作用。
试验例2本发明细胞蛋白提取物对UVB辐射损伤皮肤的修复作用研究
3D皮肤模型：商购于EpiKutis。
模型培养液：DMEM基础培养基。
VE工作液：吸取0.5g VE原液，将其溶于10mL无水乙醇中，配制成5％的母液；吸取100μL 5％母液，将其溶于10mL模型培养液中，配制成0.05％的VE工作液。
在6孔板中加入0.9mL模型培养液，将3D皮肤模型转移到6孔板中，标注测试编号。
空白对照(BC)：皮肤模型不做任何处理，将其置于CO2培养箱(37℃、5％CO2)中孵育48h；
阴性对照(NC)：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；加入25μL 0.2％的SLS工作液，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
阳性对照(PC)：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；添加25ul的0.05％的VE工作液，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h；
试验组：在皮肤模型表面辐照剂量为600mJ/cm2的UVB后，将其置于CO2培养箱(37℃、5％CO2)中孵育24h；添加25ul 1％的细胞蛋白提取物溶液(将本发明实施例4制得的细胞蛋白提取物冻干粉用生理盐水制成1％的溶液)，再将其置于CO2培养箱(37℃、5％CO2)中孵育24h。
孵育48h后，用PBS溶液洗涤并清除皮肤模型内外残留液体。用4％的多聚甲醛固定处理24h后，将模型环切取下，经H&E染色处理后观察，结果见图2。检测UVB辐射损伤皮肤的活性氧(IOD)、超氧化物歧化酶、丙二醛(MDA)水平，结果见图3-图5。本发明的细胞蛋白提取物对UVB辐射损伤皮肤具有明显的修复作用。
试验例3本发明细胞蛋白提取物对关节损伤及软骨损伤的修复作用研究
将普通饲养体重为47kg的12个月龄雄性山羊行全麻后，在其左内髁取正中切口8cm，髌腱内侧缘分离切开至关节,暴露股骨内髁及内侧胫骨平台,关节软骨表面光滑，在股骨内髁用3mm球钻做0.3×0.3×0.6cm(长×宽×深)大小的软骨损伤创面后，缝合切口(见图6)。在其右外平台取正中切口8cm,逐层分离,于髌腱外侧进入关节囊,暴露股骨外髁及胫骨外侧平台，见软骨完整光滑,用3mm球钻在平台外侧做0.6×0.3×0.3cm(长×宽×深)大小的软骨损伤创面，缝合切口(见图7)。
每周单侧给予山羊膝关节腔内注射2.5ml实施例6的细胞蛋白提取物冻干制剂溶液(将实施例6的细胞蛋白提取物冻干制剂用生理盐水制成2.4％的溶液)，连续注射三周。每天早晚各观察一次，结果见表1。
观察第49天后，麻醉处死试验羊，取材做软骨切片(见图6-7)。
本发明的细胞蛋白提取物具有优异地修复关节损伤作用，且安全有效。
以上对本发明具体实施方式的描述并不限制本发明，本领域技术人员可以根据本发明作出各种改变或变形，只要不脱离本发明的精神，均应属于本发明权利要求保护的范围。

Claims (10)

  1. 一种具有细胞修复功效的细胞蛋白提取物，其制备包括如下步骤：
    (1)将密度为1.0×106个/mL-5.0×107个/mL的间充质传代细胞置于含有DMEM/F12 40-50％、RPMI 1640 40-50％、牛血清蛋白(BSA)0.1-2％、表皮细胞生长因子(EGF)1-15ug/mL、成纤维细胞生长因子(FGF)1-15ug/mL、胰岛素转铁蛋白1-15ug/mL、复方氨基酸(18AA)0.01-0.1％和2-10μmol/L应激物的培养基中，再将其置于37.0℃±0.5℃、5％±1.0％CO2条件下培养10min-14h后，分离，洗涤，收集细胞，其中，所述的应激物选自化合物1-16的任一种或其组合，

    (2)将收集细胞按照密度为5.0×106个/mL-1.0×107个/mL分散于溶剂中，再将其置于2℃-8℃条件下超声处理，制得细胞裂解液，其中，所述溶剂选自选自生理盐水、5％葡萄糖溶液、磷酸盐缓冲液(PBS)、TBPS缓冲液、TBST缓冲液、Tris缓冲液的任一种或其组合；
    (3)将步骤(2)制得的细胞裂解液分离后，所得的分离液依次经0.45um、0.22um滤膜过滤，即得。
  2. 如权利要求1所述的蛋白提取物，步骤(1)的培养基中含有DMEM/F12 42-45％、RPMI 1640 42-45％、牛血清蛋白(BSA)0.5-1.5％、表皮细胞生长因子(EGF)5-10ug/mL、成纤维细胞生长因子(FGF) 5-10ug/mL、胰岛素转铁蛋白5-10ug/mL、复方氨基酸(18AA)0.02-0.05％和3-8μmol/L的应激物。
  3. 如权利要求1-2任一项所述的蛋白提取物，步骤(1)的间充质传代细胞密度为2.0×106-2.0×107个/mL，优选为5.0×106-1.0×107个/mL。
  4. 如权利要求1-3任一项所述的蛋白提取物，步骤(1)所述的分离选自离心、过滤的任一种或其组合，其中，所述离心条件为1000-2000rpm*3-15min，优选为1200rpm-1500rpm*5-10min。
  5. 如权利要求1-4任一项所述的蛋白提取物，步骤(2)的超声条件为：在2℃-8℃、25kHZ、360W条件下工作3s再间隙1s，超声处理1-5min。
  6. 如权利要求1-5任一项所述的蛋白提取物，在步骤(3)收集到的滤液中加入冻干保护剂，制得冻干制剂，其中，所述冻干保护剂选自甘露醇、山梨糖醇、右旋糖酐、甘油、蔗糖、海藻糖、葡萄糖、乳糖、麦芽糖、葡聚糖、三辛酸甘油酯(HES)、聚乙二醇、乙烯乙二烯、磷酸盐、醋酸盐、柠檬酸盐、山梨醇、淀粉中的任一种或其组合。
  7. 如权利要求1-6任一项所述的蛋白提取物，以质量百分比计，所述冻干制剂中含有冻干保护剂0.7-7％，优选为1-5％。
  8. 如权利要求1-7任一项所述的蛋白提取物，所述冻干制剂pH6-8，优选为pH7-7.5。
  9. 一种细胞修复组合物，所述组合物由如权利要求1-8任一项所述的具有细胞修复功效的细胞蛋白提取物和药学上可接受的载体组 成。
  10. 如权利要求1-8任一项所述的具有细胞修复功效的细胞蛋白提取物或如权利要求9所述的组合物用于制备细胞修复、关节修复、软骨修复、皮肤损伤细胞修复、神经损伤细胞修复、脏器损伤修复、肺纤维化修复、肝损伤修复、肾损伤修复、卵巢修复、抗克罗恩病、抗亚健康、抗衰的任一种的药物或制品中的应用。
PCT/CN2023/073566 2022-01-28 2023-01-28 一种细胞修复蛋白提取物及其制备方法和其应用 WO2023143519A1 (zh)

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