Culture method for improving proliferation and migration potential of mesenchymal stem cells by using traditional Chinese medicine composition

Abstract

The invention discloses a culture method for improving proliferation and migration potential of mesenchymal stem cells by using a traditional Chinese medicine composition, which adopts the traditional Chinese medicine composition taking red dates and cubilose as main components, and comprises 25 g of red dates, 25 g of cubilose, 10 g of tremella, 10 g of cranberries, 5 g of acerola cherries, 5 g of hawthorn, 5 g of astragalus membranaceus, 5 g of rehmannia, 5 g of donkey-hide gelatin and 5 g of II-type collagen cartilage powder. The preparation product is added with water, ground, filtered and then added into a mesenchymal stem cell culture medium to be cultured, and it is found that the water extract of the traditional Chinese medicine preparation can enhance the proliferation and migration potential of mesenchymal stem cells and increase the expression of anti-inflammatory factors in exosomes. The culture method of the mesenchymal stem cells is improved, so that the cell proliferation and migration potential of the mesenchymal stem cells is enhanced, the anti-inflammatory capacity of the mesenchymal stem cell exosome is enhanced, and the effect of the mesenchymal stem cells on treating the ovarian hypofunction is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of mesenchymal stem cell culture, and relates to a culture method for improving the proliferation and migration potential of mesenchymal stem cells by utilizing a traditional Chinese medicine composition. Background Art

[0002] Stem cells are one of the hot topics in life science research in the 21st century. With the rapid development of regenerative medicine and tissue engineering, stem cells have shown great potential in the field of tissue and organ repair and regeneration. Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells with the potential for self-renewal, high proliferation and multidirectional differentiation. Compared with embryonic stem cells, MSCs involve fewer ethical issues and show great application prospects in cell replacement therapy and regenerative medicine.

[0003] At present, the commonly used method for culturing mesenchymal stem cells is mainly to use T4 culture medium combined with serum for culturing. This method results in inconsistent quality of mesenchymal stem cells cultured and produced in different batches, resulting in inconsistent functions of mesenchymal stem cells in different batches.

[0004] There are related studies. For example, Chinese patent publication number CN117427114A provides a traditional Chinese medicine composition for preparing bone marrow mesenchymal stem cells (20-40 parts of Polygonum cuspidatum, 20-40 parts of Astragalus, 20-40 parts of Epimedium, 20-40 parts of Morinda officinalis, 20-40 parts of Eriobotrya japonica, 5-25 parts of Ligusticum chuanxiong, 20-40 parts of Rehmannia glutinosa, and 3-15 parts of Inula japonica). Mice were gavaged with the traditional Chinese medicine combination for 2 days, and then their bone marrow-derived mesenchymal stem cells (BMMSCs) were extracted. The results showed that the BMMSCs of the gavage group mice had stronger proliferation and migration potential than those of the control group mice.

[0005] Chinese Patent Publication No. CN115896010A discloses that by adding the compound β-nicotinamide mononucleotide or coenzyme Q10 to a conventional mesenchymal stem cell DMEM serum-free culture medium, the results showed that the proliferation and migration abilities of the mesenchymal stem cells were enhanced.

[0006] The proliferation and migration abilities of mesenchymal stem cells are their key functions for treating diseases, but there are few methods to enhance the cell proliferation and migration abilities of mesenchymal stem cells. That is, the unstable batch-to-batch quality and insufficient proliferation and migration abilities of mesenchymal stem cells restrict the clinical application of mesenchymal stem cells. Summary of the invention

[0007] In view of the problems of unstable batch quality and insufficient proliferation and migration ability of mesenchymal stem cells in the prior art, the present invention provides a culture method for improving the proliferation and migration potential of mesenchymal stem cells by using a traditional Chinese medicine composition, which solves the problem of insufficient proliferation and migration ability of conventionally cultured mesenchymal stem cells. By adding a culture medium containing water extracts of traditional Chinese medicine preparations (25g red dates, 25g bird's nests, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, and 5g type II collagen cartilage powder) to culture mesenchymal stem cells, the proliferation ability of mesenchymal stem cells can be increased by 53%, and the migration ability and the expression of anti-inflammatory related genes in exosomes can be improved, thereby promoting the effect of treating ovarian dysfunction.

[0008] The present invention uses a Chinese medicine composition with red dates and bird's nest as main ingredients, including 25g red dates, 25g bird's nest, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, and 5g type II collagen cartilage powder, and makes a Chinese medicine preparation product after ultra-micro wall breaking. The present invention grinds and filters the Chinese medicine preparation with water to make a Chinese medicine preparation water extract, and adds it to the mesenchymal stem cell culture medium for culture. It is found that the culture of the Chinese medicine preparation water extract can increase the cell proliferation potential of mesenchymal stem cells by 53% compared with conventionally cultured mesenchymal stem cells, and promotes its migration potential, and increases the expression of anti-inflammatory factors in exosomes. The present invention improves the culture method of mesenchymal stem cells to enhance the proliferation and migration potential of mesenchymal stem cells, and enhances the anti-inflammatory ability of mesenchymal stem cell exosomes, and increases the effect of mesenchymal stem cells in treating ovarian dysfunction.

[0009] The purpose of the present invention is achieved through the following technical solutions: A method for culturing mesenchymal stem cells using a Chinese medicine composition to improve their proliferation and migration potential comprises the following steps: S1: Ultra-micronized wall breaking of Chinese herbal medicines: S1-1: Place the medicinal materials (25g red dates, 25g bird's nest, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, 5g type II collagen cartilage powder) in a hammer mill and grind the Chinese medicinal materials into coarse particles with a diameter of about 1 cm; S1-2: The crushed coarse particles were placed in a DZG-1.5 multifunctional Chinese patent medicine sterilizer for sterilization. After sterilization, the medicine was placed in a CT-CI hot air circulation oven for 4 hours of drying at 80°C. The moisture content of the dried medicine particles was 5%. S1-3: After the drug particles are dried, they are added to a BFM-6B1 ultrafine pulverizer, the temperature around the grinding cylinder is set to -18°C, and the drug powder is taken out after 35 minutes; S2: Tableting of Chinese herbal medicines: The drug powder obtained by ultra-micro-wall breaking is added, and the weight of the tablet is set to 1 g, the diameter is 2 cm, the thickness is 0.8 cm, the positive pressure is greater than 250 N, and the side pressure is greater than 200 N. The raw material is directly tableted to obtain a sheet-like Chinese herbal medicine preparation; S3: Mesenchymal stem cell recovery: Turn on the constant temperature water bath and preheat it to 37°C, turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; Check the cell freezing location, take out the cells from the liquid nitrogen tank, soak them in a 37°C constant temperature water bath for 2 min to revive, transfer all the contents of the cryopreserved tube to a 15 ml centrifuge tube, add 4 ml MSC-T4 complete medium (MSC-T4 + 5% UltraGRO + 1% L-glutamine, vt%) to resuspend, centrifuge at 300g for 5 min, remove the supernatant, and resuspend with 1 ml MSC-T4 complete medium. Take a T75 culture flask and add 0.89×10 4 / cm 2 Add the cell suspension to the culture flask at an inoculation density of 100 μl, add complete medium to each flask to 12 ml, shake well and transfer the cells to a carbon dioxide (CO2) incubator; S4: Culture and subculture: Turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; Take out the culture flask from the incubator, pour out the culture medium in the flask, pipette 5 ml of phosphate buffered saline (D-PBS) into the flask, shake the flask gently to rinse it, and then pipette the rinse solution into the waste liquid bottle; pipette 2 ml of recombinant trypsin (TrypLE) into the flask, shake the flask gently to cover the cell attachment surface of the flask with the liquid, and place it in a CO2 incubator for digestion for 3 minutes; Add 2 ml of complete medium to each culture bottle, shake to mix, transfer the cell suspension to a centrifuge tube, and rinse the culture bottle with 5 ml of D-PBS, then transfer the rinsed cell suspension to the above centrifuge tube; centrifuge at 400 g for 5 min, 9 for ascending and 8 for descending, at room temperature; After centrifugation, pour off the supernatant, pipette 2 ml of complete medium into a centrifuge tube to resuspend the cells, pipette and mix, take 100 μl of the cell suspension into a centrifuge tube (EP tube) for cell counting (dilute 10 times for counting) and viability detection; prepare culture flasks according to the number of cells; According to 0.89×10 4 / cm 2 Add the cell suspension to the culture bottle at the inoculation density, add complete medium to each bottle to 12 ml, shake well and transfer the cells to the CO2 incubator; each transfer to a new culture bottle is an increase of one generation, and repeat the culture to the fourth generation; S5: Grind the flaky Chinese herbal medicine and add 1 ml of water to dissolve it: S5-1: Grind the flaky Chinese medicinal material preparation into uniform fine powder using a mortar, add 1 ml of culture medium and continue grinding, and fully dissolve it to obtain a Chinese medicinal solvent; S5-2: Place the Chinese medicine solvent in a centrifuge and centrifuge at 10000 g for 10 min; S5-3: taking the supernatant, filtering it through a 0.22 μm filter membrane for sterilization, and obtaining a water extract of a sheet-like Chinese medicinal material preparation; S5-4: Add the aqueous extract of the Chinese herbal medicine preparation to the culture medium at a concentration of 5 μl / ml to participate in cell culture; S6: Exosome isolation and purification; S6-1: Centrifugation: The collected mesenchymal stem cell culture supernatant was centrifuged at 3000 rpm, 4°C for 10 min to remove cell residues and debris; S6-2: Add polyethylene glycol (PEG) solution: prepare a 20% PEG4000 solution (prepared with sterile water for injection); add the prepared PEG solution to the filtered MSC three-dimensional (3D) culture supernatant, mix at a ratio of 1:1, and make the final PEG concentration be 10%; S6-3: Mixing and refrigeration: Gently mix the biological fluid and PEG solution to ensure uniform mixing, and then place the mixture in a refrigerator at 4°C overnight or for 24 hours; S6-4: Centrifugation: The static mixture was centrifuged at 13000g, 4°C, for 30 min to precipitate the exosomes; S6-5: Remove supernatant: Carefully remove the supernatant, leaving the precipitated exosome pellet; S6-6: Resuspend the precipitate: resuspend all the precipitates obtained by centrifugation with an appropriate amount of physiological saline; S6-7: 0.22um membrane filtration: The exosome heavy suspension is filtered through a 0.22um membrane filter, and the final filtrate is the exosome solution; S7: Exosome identification: The number, size, and concentration of exosomes were analyzed using a Zetaview-PMX120-Z nanoparticle tracking analyzer; S8: Exosome protein detection: S8-1: Prepare the bovine serum albumin (BSA) standard system: dilute the standard sample in series using the standard diluent; S8-2: Prepare bicinchoninic acid (BCA) working solution: add 1 volume of BCA reagent B to 50 volumes of BCA reagent A, A:B=50:1, and mix thoroughly; S8-3: Take 20 μL of the standard and the sample to be tested and add them to the microplate; S8-4: Add 200 μl BCA working solution to each well and shake for 30 seconds to mix thoroughly; cover the microplate and incubate at 37°C for 30 minutes; S8-5: Cool to room temperature, detect absorbance at 540-595nm wavelength range on the microplate reader, with 562nm wavelength being the best. Calculate protein concentration based on known standard curve. Compared with normally cultured mesenchymal stem cells, 5μl / ml water extract of Chinese herbal medicine preparation increases the proliferation of mesenchymal stem cells by 50-60%.

[0010] Compared with the prior art, the present invention has the following advantages: 1. The method for cultivating mesenchymal stem cells by using a Chinese medicine composition of the present invention comprises the following steps: ultrafinely breaking the wall of the Chinese medicine composition (25 g of red dates, 25 g of bird's nest, 10 g of white fungus, 10 g of cranberry, 5 g of acerola, 5 g of hawthorn, 5 g of astragalus, 5 g of rehmannia, 5 g of donkey-hide gelatin, and 5 g of type II collagen cartilage powder) and tableting the composition to prepare a Chinese medicine preparation. The effective ingredients of the medicine are easier to dissolve, the drug release rate is fast, and the bioavailability is high.

[0011] 2. The present invention discloses a method for cultivating mesenchymal stem cells using a Chinese medicine composition to improve their proliferation and migration potential. In the process of cultivating mesenchymal stem cells, a water extract of a Chinese medicine preparation is added. It is found that compared with the mesenchymal stem cells cultured normally, 5 μl / ml of the water extract of the Chinese medicine preparation increases the proliferation of the mesenchymal stem cells by 50-60%, promotes the cell proliferation and migration ability of the mesenchymal stem cells, and enhances the anti-inflammatory ability of the exosomes of the mesenchymal stem cells.

[0012] 3. The culture method of the present invention for improving the proliferation and migration potential of mesenchymal stem cells by using a Chinese medicine composition, which uses oral administration of a water extract of a Chinese medicine preparation combined with intravenous injection of mesenchymal stem cells to treat a rat model of ovarian dysfunction, has better effects than single mesenchymal stem cell treatment and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0014] Figure 1 This is a graph showing the effects of different concentrations of water extracts of Chinese herbal medicine preparations on the proliferation capacity of mesenchymal stem cells in the experimental examples of the present invention; Figure 2 This is a graph showing the ability of the water extract of the Chinese herbal medicine preparation in promoting the migration of mesenchymal stem cells in the experimental example of the present invention; Figure 3 A graph showing the expression levels of proliferation and migration-related genes in mesenchymal stem cells cultured with water extracts of Chinese medicinal materials preparations at different concentrations in the experimental examples of the present invention; Figure 4 This is a diagram of the identification of exosomes of mesenchymal stem cells cultured with water extracts of traditional Chinese medicine preparations in the experimental examples of the present invention; Figure 5 This is a graph showing the expression of IL-4 and IL-10 proteins in exosomes of mesenchymal stem cells cultured with water extracts of traditional Chinese medicine preparations in the experimental examples of the present invention; Figure 6 This is a graph showing changes in the levels of testosterone (T), estradiol (E2), progesterone (P), luteinizing hormone (LH), prolactin (PRL), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH) in the serum of rats in the experimental examples of the present invention. DETAILED DESCRIPTION

[0015] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified. The quantitative tests in the following examples are all repeated three times.

[0016] Example: A method for culturing mesenchymal stem cells using a Chinese medicine composition to improve their proliferation and migration potential comprises the following steps: S1: Ultra-micronized wall breaking of Chinese herbal medicines: S1-1: Place the medicinal materials (25g red dates, 25g bird's nest, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, 5g type II collagen cartilage powder) in a hammer mill and grind the Chinese medicinal materials into coarse particles with a diameter of about 1 cm; S1-2: The crushed coarse particles are placed in a DZG-1.5 multifunctional Chinese patent medicine sterilizer for sterilization. After sterilization, the medicine is placed in a CT-CI hot air circulation oven for 4 hours of drying at 80°C. The moisture content of the dried medicine particles is about 5%; S1-3: After the drug particles are dried, they are added to a BFM-6B1 ultrafine pulverizer, the temperature around the grinding cylinder is set to -18°C, and the drug powder is taken out after 35 minutes; S2: Tableting of Chinese herbal medicines: Add the powder obtained by ultra-micro-wall breaking, set the weight of the tablet to 1 g, the diameter to 2 cm, the thickness to 0.8 cm, the positive pressure to > 250 N, the side pressure to > 200 N, and directly compress the raw materials to obtain a sheet-like Chinese herbal medicine preparation; S3: Mesenchymal stem cell recovery: Turn on the constant temperature water bath and preheat to 37°C; turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; check the cell freezing position, take out the cells from the liquid nitrogen tank, soak them in a 37°C constant temperature water bath for 2 minutes to revive, transfer all the contents of the cryopreserved tube to a 15ml centrifuge tube, add 4ml MSC-T4 complete medium (MSC-T4+5% UltraGRO+1% L-glutamine, vt%) to resuspend, centrifuge at 300g for 5 minutes, remove the supernatant, and resuspend with 1ml MSC-T4 complete medium, take a T75 culture flask, and adjust the temperature according to 0.89×10 4 / cm 2 Add the cell suspension to the culture flask at an inoculation density of 100 μl, add complete medium to each flask to 12 ml, shake well and transfer the cells to a carbon dioxide (CO2) incubator; S4: Culture and subculture: Turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; take out the culture bottle from the incubator, pour out the culture medium in the bottle, pipette 5ml of phosphate buffered saline (D-PBS) into the culture bottle, gently shake the culture bottle to rinse, and then pipette the rinse solution into the waste liquid bottle; pipette 2ml of recombinant trypsin (TrypLE) into the culture bottle, gently shake the culture bottle to make the liquid cover the cell attachment surface of the culture bottle, and put it into the CO2 incubator for digestion for 3min; add 2ml of complete culture medium to each culture bottle, shake to mix, transfer the cell suspension into a centrifuge tube, and pipette 5ml The culture flasks were rinsed with D-PBS in turn, and then the cell suspension after rinsing was transferred to the above centrifuge tubes; centrifuged at 400g×5min, 9 up and 8 down, at room temperature; after centrifugation, the supernatant was poured out, 2ml of complete culture medium was taken into the centrifuge tube to resuspend the cells, and the mixture was mixed by pipetting, and about 100μl of cell suspension was taken into the centrifuge tube (EP tube) for cell counting (diluted 10 times for counting) and viability detection; culture flasks were prepared according to the number of cells; according to 0.89×10 4 / cm 2 Add the cell suspension to the culture bottle at the inoculation density, add complete medium to each bottle to 12 ml, shake well and transfer the cells to the CO2 incubator; each transfer to a new culture bottle is an increase of one generation, and repeat the culture to the fourth generation; S5: Grind the flaky Chinese herbal medicine and add 1 ml of water to dissolve it: S5-1: Use a mortar to grind the Chinese herbal medicine into a uniform fine powder, add 1 ml of culture medium and continue grinding to fully dissolve it; S5-2: Place the Chinese medicine solvent in a centrifuge and centrifuge at 10000 g for 10 min; S5-3: taking the supernatant, filtering it through a 0.22 μm filter membrane for sterilization, and obtaining a water extract of a sheet-like Chinese medicinal material preparation; S5-4: Add the aqueous extract of the Chinese herbal medicine preparation to the culture medium at a concentration of 5 μl / ml to participate in cell culture; S6: Exosome isolation and purification; S6-1: Centrifugation: The collected mesenchymal stem cell culture supernatant was centrifuged at 3000 rpm, 4°C for 10 min to remove cell residues and debris; S6-2: Add polyethylene glycol (PEG) solution: prepare a 20% PEG4000 solution (prepared with sterile water for injection); add the prepared PEG solution to the filtered MSC three-dimensional (3D) culture supernatant, mix at a ratio of 1:1, and make the final PEG concentration be 10%; S6-3: Mixing and refrigeration: Gently mix the biological fluid and PEG solution to ensure uniform mixing, and then place the mixture in a refrigerator at 4°C overnight or for 24 hours; S6-4: Centrifugation: The static mixture was centrifuged at 13000g, 4°C, for 30 min to precipitate the exosomes; S6-5: Remove supernatant: Carefully remove the supernatant, leaving the precipitated exosome pellet; S6-6: Resuspend the precipitate: resuspend all the precipitates obtained by centrifugation with an appropriate amount of physiological saline; S6-7: 0.22um membrane filtration: The exosome heavy suspension is filtered through a 0.22um membrane filter, and the final filtrate is the exosome solution; S7: Exosome identification: The number, size, and concentration of exosomes were analyzed using a Zetaview-PMX120-Z nanoparticle tracking analyzer; S8: Exosome protein detection: S8-1: Prepare the bovine serum albumin (BSA) standard system: dilute the standard sample in series using the standard diluent; S8-2: Prepare bicinchoninic acid (BCA) working solution: add 1 volume of BCA reagent B to 50 volumes of BCA reagent A, A:B=50:1, and mix thoroughly; S8-3: Take 20 μL of the standard and the sample to be tested and add them to the microplate; S8-4: Add 200 μl BCA working solution to each well and shake for 30 seconds to mix thoroughly; cover the microplate and incubate at 37°C for 30 minutes; S8-5: Cool to room temperature, measure the absorbance at a wavelength range of 540-595 nm on a microplate reader, with 562 nm being the best wavelength, and calculate the protein concentration based on a known standard curve; S9: Cell Counting Kit-8 (CCK-8) Cell Proliferation Assay: The cell suspension obtained by digestion of mesenchymal stem cells was inoculated into a 96-well cell culture plate at 100 μl / well. The plate was placed in an incubator for culture. When the cells were confluent to 60%-70%, the culture medium containing different concentrations of water extracts of Chinese herbal medicine preparations was replaced and cultured for 24 h. After the treatment time was reached, the cells were subjected to color development reaction using CCK-8 reagent for 1 h (10 μl was added to each well, and care was taken not to generate bubbles in the wells), and the absorbance at 450 nm was measured using an enzyme reader. S10: Real-time fluorescence quantitative PCR (qPCR) detection: S10-1: RNA extraction method: The cell suspension obtained by digestion of mesenchymal stem cells was inoculated into a 6-well cell culture plate, 2 ml / well, and the plate was placed in an incubator for culture. When the cells were confluent to 60%-70%, the medium containing different concentrations of water extracts of Chinese herbal medicine preparations was replaced and cultured for 24 h. After the treatment time was reached, the supernatant was removed, and the cells were rinsed twice with D-PBS. Then, the cells were digested from the culture plate with 0.25wt% trypsin, centrifuged at 300g for 5 min, and washed once with pre-cooled PBS; each 1.0×10 7 Add 1 ml of the novel total RNA extraction reagent (TRIzol) to the cells; transfer the cell lysate to a 1.5 ml RNase-free EP tube and let it stand for 5 min; add 200 μl of chloroform to each tube, mix thoroughly and let it stand at room temperature for 10 min to completely dissociate the nucleoprotein complex; Centrifuge at 4°C, 12000g for 15 min; after centrifugation, transfer the upper aqueous phase to the new EP tube; add 0.5 ml of isopropanol and leave at room temperature for 10 min; Centrifuge at 12000g for 10 min at 4°C, remove the supernatant, and wash the RNA precipitate once with 1 ml of 75% ethanol; Centrifuge at 12000 g for 5 min at 4°C and remove the supernatant; wash the RNA precipitate once with 1 ml of anhydrous ethanol; Centrifuge at 12000g for 5 min at 4°C and discard the supernatant; air-dry or vacuum-dry the RNA precipitate for 5-10 min; RNA was dissolved in 30 μl of deionized water treated with diethylpyrocarbonate (DEPC); sample concentration and purity were determined by spectrophotometric analysis; S10-2: RNA reverse transcription: Take out gDNA remover, 10x gDNA remover Buffer, and RNase-free water from the reverse transcription kit, place them on ice to melt, and centrifuge for a few seconds before use; take eight qPCR tubes and prepare the reverse transcription reaction according to the table below:

[0017] Then gently mix with a pipette tip, centrifuge briefly, incubate at 42°C for 2 min, and then incubate at 60°C for 5 min; quickly cool the mixture on ice, centrifuge briefly, and add the following components:

[0018] Finally, mix gently with a pipette tip, centrifuge briefly, warm the reaction mixture at 25°C for 10 min, then heat at 55°C for 15 min, and then heat at 85°C for 5 min to inactivate the enzyme, and store at 4°C; the obtained cDNA reaction solution can be directly used as a template for fluorescence quantitative detection; perform reverse transcription reaction according to the following reaction conditions; S10-3: Fluorescence quantitative PCR: Design qPCR primers for genes cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4), sex-determining region Y-box 2 (SOX2), octamer-binding transcription factor (OCT4), chemokine receptor-4 (CXCR4), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and integrin subunit β1 (ITGB1):

[0019] Add the following reaction mixture to a PCR tube in an ice bath: 5µl 2×TSINGKE® Master qPCR Mix (SYBR Green I with UDG), 0.4µl forward PCR primer, 0.4µl reverse PCR primer, 1 µl cDNA reaction solution, 3.2 µl double distilled water (ddH2O); gently mix and centrifuge for 3-5 seconds, put the reaction mixture into the qPCR instrument, and the reaction program is: 50℃ 2 min, 1 cycle; 95℃ 2 min, 1 cycle; 95℃ 15s, 60℃ 30s, 40 cycles; after the reaction, take out the PCR tube and calculate 2 according to the absorbance (CT) value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and each experimental group. -∆∆Ct , the relative expression of genes in the treatment group was obtained; S11: Cell migration assay: S11-1: Human ovarian granulosa cells (KGN cells) were selected as a cell disease model of ovarian dysfunction and plated in the lower chamber of a 24-well Transwell plate. KGN cells were treated with 1.6 mM H2O2 for 2 h, then H2O2 was removed and cultured for another 24 h. S11-2: mesenchymal cells treated with traditional Chinese medicine were added to the upper layer of the Transwell plate and cultured for 24 h; S11-3: Remove the supernatant, take out the upper chamber of the Transwell plate, remove the culture medium, fix the cells with 4% paraformaldehyde fixative for 30 min, discard the fixative, wash the inside and outside of the chamber with PBS once, add 600 μl of crystal violet staining solution to the clean wells of the 24-well plate, and place the chamber in the staining for 10 min; S11-4: After proper air drying, observe the results under a microscope.

[0020] Experimental Example 1: The cultured cells were inoculated into 96-well plates at 10,000 cells / well and cultured overnight. When the cells grew to 60%-70%, water extracts of Chinese herbal medicine preparations were added to the blank control group (0 μl / ml), low-dose group (1 μl / ml), medium-dose group (5 μl / ml) and high-dose group (10 μl / ml). The culture was continued for 24 hours and the proliferation ability of mesenchymal stem cells was detected by CCK-8 experiment. The results are shown in Figure 1 As shown, the optical density (OD) values ​​of the medium-dose group and the low-dose group were higher than that of the blank control group, that is, compared with the blank control group without the addition of the water extract of Chinese herbal medicine preparation, the low-dose group (1 μl / ml) of the water extract of Chinese herbal medicine preparation increased the proliferation of mesenchymal stem cells by 16%, and the medium-dose (5 μl / ml) of the water extract of Chinese herbal medicine preparation increased the proliferation of mesenchymal stem cells by 53%. The medium-dose group had a higher cell proliferation-promoting effect than the low-dose group, while the OD value of the high-dose group was significantly lower than that of the blank control group, indicating that the addition of low-dose and medium-dose water extracts of Chinese herbal medicine preparations can promote the proliferation ability of mesenchymal stem cells, and the promotion effect is better at the medium dose. In the process of mesenchymal stem cell culture, the addition of high-dose water extracts of Chinese herbal medicine preparations may have certain cytotoxicity.

[0021] Figure 1 Effects of different concentrations of aqueous extracts of Chinese herbal medicines on the proliferation capacity of mesenchymal stem cells.

[0022] Embodiment 2: Cell migration assay In Example 1, we found that 10 μl / ml of Chinese herbal medicine water extract had certain cytotoxicity in mesenchymal stem cell culture, so we removed the 10 μl / ml concentration group in the Transwell cell migration experiment, that is, in the design groups of blank control group (0 μl / ml), low-dose group (1 μl / ml) and high-dose group (5 μl / ml), the ovarian dysfunction cell disease model human ovarian granulosa cells (KGN cells) damaged by hydrogen peroxide (H2O2) were plated on the lower layer of 24-well Transwell plate, and 2×10 4 The cells were added to the upper layer of the Transwell plate, and after culturing for 24 hours, crystal violet staining solution was added to observe the cell migration ability. Figure 2 As shown, mesenchymal stem cells were found in the lower layer of the Transwell plate in both the low-dose and high-dose groups, and more mesenchymal stem cells appeared in the lower layer of the Transwell plate in the high-dose group than in the low-dose group, indicating that water extracts of Chinese herbal medicine preparations can promote the migration of mesenchymal stem cells, and the migration ability of mesenchymal stem cells in the high-dose group is stronger than that in the low-dose group.

[0023] Figure 2 Aqueous extracts of Chinese herbal medicines promote the migration ability of mesenchymal stem cells.

[0024] Embodiment 3: The mRNA expressions of proliferation-related genes (CCND1, CDK4, SOX2, and OCT4) and migration-related genes (CXCR4, MMP2, MMP9, and ITGB1) in mesenchymal stem cells of the high-dose group (5 μl / ml), the low-dose group (1 μl / ml), and the blank control group (0 μl / ml) were detected by RT-qPCR. Figure 3 As shown, it was found that these genes were higher in both the high-dose group and the low-dose group than in the blank control group, and the expression was higher in the high-dose group, indicating that high-dose (5μl / ml) of water extract of Chinese herbal medicine preparation can better promote the proliferation and migration ability of mesenchymal stem cells.

[0025] Figure 3 Expression levels of proliferation- and migration-related genes in mesenchymal stem cells cultured with different concentrations of aqueous extracts of Chinese medicinal materials.

[0026] Embodiment 4: After separation and purification of exosomes, identification was performed. Figure 4As shown in the results, it was found that the morphology, concentration and particle size of mesenchymal stem cell exosomes cultured with 5 μl / ml Chinese herbal medicine water extract did not change significantly. Further protein detection revealed that the expression of anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10) in exosomes increased significantly. The results are shown in Figure 5 As shown. This indicates that the exosomes of mesenchymal stem cells cultured with 5 μl / ml water extract of Chinese herbal medicine preparations have a stronger immunomodulatory effect.

[0027] Figure 4 Identification of exosomes from mesenchymal stem cells cultured with aqueous extracts of traditional Chinese medicine preparations; Figure 5 Expression of IL-4 and IL-10 proteins in exosomes of mesenchymal stem cells cultured with aqueous extracts of traditional Chinese medicine preparations.

[0028] Example 5 Forty female Sprague-Dawley (SD) rats were randomly divided into a combined treatment group (ovarian hypofunction modeling + intragastric administration of water extract of Chinese herbal medicine preparation + tail vein injection of mesenchymal stem cells), an mesenchymal stem cell treatment group (ovarian hypofunction modeling + intragastric administration of normal saline + tail vein injection of mesenchymal stem cells), a model group (ovarian hypofunction modeling + intragastric administration of normal saline + tail vein injection of normal saline) and a blank control group (intergastric administration of normal saline + tail vein injection of normal saline), with 10 female rats in each group. After SD rats were adaptively fed for one week, SD rats were induced to have an ovarian hypofunction model by paclitaxel, and the administration method was tail vein infusion, once every two weeks, for a total of 2 times. On the second day after successful modeling, the combined treatment group and the mesenchymal stem cell treatment group received a single tail vein injection of mesenchymal stem cells, and the model group and the blank control group received a single tail vein injection of normal saline. The rats in the combined treatment group were intragastricly administered with water extract of Chinese herbal medicine preparations every day for 7 consecutive days, and the mesenchymal stem cell treatment group, the model group and the blank control group were intragastricly administered with normal saline every day for 7 consecutive days. After the experiment, blood was collected from the rats’ orbits and centrifuged at 3000 r / min for 15 min to obtain serum. The changes in the levels of testosterone (T), estradiol (E2), progesterone (P), luteinizing hormone (LH), prolactin (PRL), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH) in the rats’ serum were observed before administration, on the 3rd day, and on the 7th day. Figure 6 As shown, there was no significant difference in hormone P among the groups. T, LH, and FSH in the model group increased significantly, and E2, PRL, and AMH decreased significantly. Both mesenchymal stem cells and combined treatment stabilized the changes in hormone levels, and the combined treatment group had a better effect.

[0029] Figure 6Changes in the levels of testosterone (T), estradiol (E2), progesterone (P), luteinizing hormone (LH), prolactin (PRL), follicle-stimulating hormone (FSH), and anti-Mullerian hormone (AMH) in rat serum; Note: * indicates that the group is significantly different from Day 0 (P<0.05).

[0030] Results and Discussion: The present application discloses a culture method for improving the proliferation and migration potential of mesenchymal stem cells by using a Chinese medicine composition. The Chinese medicine composition mainly comprises red dates and bird's nests, including 25g red dates, 25g bird's nests, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, and 5g type II collagen cartilage powder. The Chinese medicine preparation is made into a Chinese medicine preparation after ultra-micro wall breaking and tableting. The Chinese medicine preparation is then ground and filtered with water to make a Chinese medicine preparation water extract, which is then added to a mesenchymal stem cell culture medium for culture. It is found that compared with the mesenchymal stem cells cultured normally, 1 μl / ml of the Chinese medicine preparation water extract increases the proliferation of mesenchymal stem cells by 16%, 5 μl / ml of the Chinese medicine preparation water extract increases the proliferation of mesenchymal stem cells by 53%, and 10 μl / ml of the Chinese medicine preparation water extract makes the proliferation ability of mesenchymal stem cells lower than that of normal culture. This indicates that the addition of 1 μl / ml and 5 μl / ml of Chinese herbal medicine water extract can promote the proliferation of mesenchymal stem cells, and the promoting effect is better at medium doses, while 10 μl / ml of Chinese herbal medicine water extract may have certain cytotoxicity.

[0031] Cell migration experiments showed that 1 μl / ml and 5 μl / ml Chinese herbal medicine water extracts could promote the migration of mesenchymal stem cells, and 5 μl / ml Chinese herbal medicine water extracts had a better effect on promoting the migration of mesenchymal stem cells. RT-qPCR detection found that mesenchymal stem cells cultured with 5 μl / ml and 1 μl / ml Chinese herbal medicine water extracts had higher mRNA expressions of proliferation-related genes (CCND1, CDK4, SOX2 and OCT4) and migration-related genes (CXCR4, MMP2, MMP9, ITGB1) than those cultured with conventional mesenchymal stem cells, and 5 μl / ml Chinese herbal medicine water extracts had a higher promoting effect than 1 μl / ml Chinese herbal medicine water extracts. There was no significant change in the morphology, concentration and particle size of exosomes of mesenchymal stem cells cultured with 5 μl / ml Chinese herbal medicine water extracts.

[0032] Protein detection revealed that the expression of anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10) in exosomes was significantly increased. This indicates that 5 μl / ml water extract of Chinese herbal medicine preparations cultured mesenchymal stem cell exosomes have a stronger immunomodulatory effect. The use of Chinese herbal medicine preparation water extract gavage combined with tail vein injection of mesenchymal stem cells to treat ovarian dysfunction model rats found that mesenchymal stem cell treatment alone or combined treatment significantly increased T, LH, FSH, and significantly decreased E2, PRL, and AMH in the model group rats. Both mesenchymal stem cell treatment alone and combined treatment improved hormone levels, and the combined treatment had a better effect.

[0033] At present, the commonly used method for culturing mesenchymal stem cells is mainly to use T4 culture medium combined with serum for culturing. The method obtains inconsistent quality of mesenchymal stem cells cultured and produced in different batches, resulting in inconsistent functions of mesenchymal stem cells in different batches, wherein the cell proliferation and migration ability of mesenchymal stem cells are the key functions for treating diseases, but there are few methods to enhance the cell proliferation and migration ability of mesenchymal stem cells, that is, the batch-to-batch quality instability and proliferation and migration ability of mesenchymal stem cells are insufficient, which restricts the clinical application of mesenchymal stem cells. The present invention combines mesenchymal stem cell treatment with a Chinese medicine composition with red dates and bird's nest as the main ingredients to enhance the cell proliferation and migration potential of mesenchymal stem cells, and enhances the anti-inflammatory ability of mesenchymal stem cell exosomes, increasing the effect of mesenchymal stem cells in treating ovarian dysfunction. Strengthen the batch-to-batch quality stability of mesenchymal stem cells and promote the clinical application of mesenchymal stem cells.

[0034] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for cultivating mesenchymal stem cells by using a Chinese medicine composition to improve their proliferation and migration potential, characterized in that: The following steps are involved: S1: Ultra-micronized wall breaking of Chinese herbal medicines: S1-1: Place the medicinal materials in a hammer mill for processing and crush the Chinese medicinal materials into coarse particles with a diameter of 1 cm; The medicinal materials are composed of 25g red dates, 25g bird's nest, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, and 5g type II collagen cartilage powder; S1-2: The crushed coarse particles were placed in a DZG-1.5 multifunctional Chinese patent medicine sterilizer for sterilization. After sterilization, the medicine was placed in a CT-CI hot air circulation oven for 4 hours of drying at 80°C. The moisture content of the dried medicine particles was 5%. S1-3: After the drug particles are dried, they are added to a BFM-6B1 ultrafine pulverizer, the temperature around the grinding cylinder is set to -18°C, and the drug powder is taken out after 35 minutes; S2: Tableting of Chinese herbal medicines: The drug powder obtained by ultra-micro-wall breaking is added, and the weight of the tablet is set to 1 g, the diameter is 2 cm, the thickness is 0.8 cm, the positive pressure is greater than 250 N, and the side pressure is greater than 200 N. The raw material is directly tableted to obtain a sheet-like Chinese herbal medicine preparation; S3: Mesenchymal stem cell recovery: Turn on the constant temperature water bath and preheat it to 37°C, turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; Check the cell freezing location, take out the cells from the liquid nitrogen tank, soak them in a 37°C constant temperature water bath for 2 min to revive, transfer all the contents of the cryopreserved tube to a 15 ml centrifuge tube, add 4 ml MSC-T4 complete medium to resuspend, centrifuge at 300 g for 5 min, remove the supernatant, and resuspend with 1 ml MSC-T4 complete medium. Take a T75 culture flask and add 0.89×10 4 / cm 2 Add the cell suspension to the culture flask at the inoculation density, add complete medium to each flask to 12 ml, shake well and transfer the cells to the CO2 incubator; S4: Culture and subculture: Turn on the clean bench, wipe the entire clean bench evenly with a sterile cloth containing 75% alcohol, and put in the required reagents and consumables; Take out the culture flask from the incubator, pour out the culture medium in the flask, pipette 5 ml of phosphate buffer into the flask, shake the flask gently to rinse it, and then pipette the rinse solution into the waste liquid bottle; pipette 2 ml of recombinant trypsin into the flask, shake the flask gently to make the liquid cover the cell attachment surface of the flask, and place it in a CO2 incubator for digestion for 3 minutes; Add 2 ml of complete medium to each culture bottle, shake to mix, transfer the cell suspension to a centrifuge tube, and rinse the culture bottle with 5 ml of D-PBS, then transfer the rinsed cell suspension to the above centrifuge tube; centrifuge at 400 g for 5 min, 9 for ascending and 8 for descending, at room temperature; After centrifugation, pour off the supernatant, pipette 2 ml of complete culture medium into a centrifuge tube to resuspend the cells, pipette and mix well, take 100 μl of the cell suspension into a centrifuge tube and dilute 10 times for cell counting and viability detection; prepare culture flasks according to the number of cells; According to 0.89×10 4 / cm 2 Add the cell suspension to the culture bottle at the inoculation density, add complete medium to each bottle to 12 ml, shake well and transfer the cells to the CO2 incubator; each transfer to a new culture bottle is an increase of one generation, and repeat the culture to the fourth generation; S5: Grind the flaky Chinese herbal medicine and add 1 ml of water to dissolve it: S5-1: Grind the flaky Chinese medicinal material preparation into uniform fine powder using a mortar, add 1 ml of culture medium and continue grinding, and fully dissolve it to obtain a Chinese medicinal solvent; The Chinese herbal medicine preparation is made of 25g red dates, 25g bird's nest, 10g white fungus, 10g cranberry, 5g acerola, 5g hawthorn, 5g astragalus, 5g rehmannia, 5g donkey-hide gelatin, and 5g type II collagen cartilage powder; S5-2: Place the Chinese medicine solvent in a centrifuge and centrifuge at 10000 g for 10 min; S5-3: taking the supernatant, filtering it through a 0.22 μm filter membrane for sterilization, and obtaining a water extract of a sheet-like Chinese medicinal material preparation; S5-4: Add the aqueous extract of the Chinese herbal medicine preparation to the culture medium at a concentration of 5 μl / ml to participate in cell culture; S6: Exosome isolation and purification; S6-1: Centrifugation: The collected mesenchymal stem cell culture supernatant was centrifuged at 3000 rpm, 4°C for 10 min to remove cell residues and debris; S6-2: Adding polyethylene glycol solution: Prepare a 20% PEG4000 solution; add the prepared PEG solution to the filtered MSC 3D culture supernatant, mix at a ratio of 1:1, and make the final PEG concentration be 10%; S6-3: Mixing and refrigeration: Gently mix the biological fluid and PEG solution to ensure uniform mixing, and then place the mixture in a refrigerator at 4°C overnight or for 24 hours; S6-4: Centrifugation: The static mixture was centrifuged at 13000g, 4°C, for 30 min to precipitate the exosomes; S6-5: Remove supernatant: Carefully remove the supernatant, leaving the precipitated exosome pellet; S6-6: Resuspend the precipitate: resuspend all the precipitates obtained by centrifugation with an appropriate amount of physiological saline; S6-7: 0.22um membrane filtration: The exosome heavy suspension is filtered through a 0.22um membrane filter, and the final filtrate is the exosome solution; S7: Exosome identification: The number, size, and concentration of exosomes were analyzed using a Zetaview-PMX120-Z nanoparticle tracking analyzer; S8: Exosome protein detection: S8-1: Prepare BSA standard system: Use standard diluent to perform gradient dilution of the standard; S8-2: Prepare BCA working solution: add 1 volume of BCA reagent B to 50 volumes of BCA reagent A, A:B=50:1, and mix thoroughly; S8-3: Take 20 μL of the standard and the sample to be tested and add them to the microplate; S8-4: Add 200 μl BCA working solution to each well and shake for 30 seconds to mix thoroughly; cover the microplate and incubate at 37°C for 30 minutes; S8-5: Cool to room temperature, detect absorbance at 540-595nm wavelength range on the microplate reader, with 562nm wavelength being the best. Calculate protein concentration based on known standard curve. Compared with normally cultured mesenchymal stem cells, 5 μl / ml water extract of Chinese herbal medicine preparation increases the proliferation of mesenchymal stem cells by 50-60%.

2. The method for cultivating mesenchymal stem cells by using a Chinese medicine composition according to claim 1, characterized in that: Add 4 ml of MSC-T4 complete medium as described in step S2 and resuspend, wherein the MSC-T4 complete medium is: MSC-T4 + 5 vt% UltraGRO + 1 vt% L-glutamine.

3. The method for cultivating mesenchymal stem cells by using a Chinese medicine composition according to claim 1, characterized in that: The 20% PEG4000 solution described in step S6-2 is prepared using sterile water for injection.

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