Induction medium for preparing human umbilical cord mesenchymal stem cell exosome and application thereof

Abstract

The invention discloses an induction culture medium for preparing human umbilical cord mesenchymal stem cell exosomes and application of the induction culture medium, and relates to the technical field of exosome culture. The additive is prepared from the following components: 0.8 to 1.5 percent of ITS, 0.03 to 0.05 percent of a luteolin extract, 0.04 to 0.06 percent of a herba taraxaci extract, 0.02 to 0.04 percent of a radix ginseng extract, 0.03 to 0.05 percent of resveratrol, 0.01 to 0.03 percent of vitamin C, 0.03 to 0.05 percent of a pimpinella thellungiana extract, 0.01 to 0.03 percent of a rhizoma dioscoreae extract and 0.01 to 0.02 percent of sodium citrate; the induction culture medium provided by the invention not only can avoid introduction of exogenous substances and reduce the pollution risk, but also can promote proliferation of stem cells and effectively induce synthesis and exosome secretion of a large number of human umbilical cord mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome culture, and in particular to an induction culture medium for preparing human umbilical cord mesenchymal stem cell exosomes and an application thereof. Background Art

[0002] Stem cells are a type of cell with self-renewal and differentiation potential, mainly including embryonic stem cells (ESCs), adult stem cells (ASCs), induced pluripotent stem cells (iPSCs) and tissue resident stem cells (Tissue resident stem cells). Mesenchymal stem cells (MSCs) are a type of adult stem cells. MSCs are derived from the early mesoderm of development and are pluripotent stem cells with strong self-renewal ability and multidirectional differentiation potential. They can differentiate into bone cells, chondrocytes and adipocytes. At present, there are many basic studies on umbilical cord mesenchymal stem cells, adipose mesenchymal stem cells and bone marrow mesenchymal stem cells. The functions and effects of the three types of MSCs have also been clearly verified. Clinical departments are gradually using MSCs as a new alternative therapy.

[0003] The modes of action of mesenchymal stem cells mainly include direct cell action and paracrine action. Currently, researchers believe that the most important mode of action of MSCs is paracrine action. Exosomes are extracellular vesicles with a diameter of about 30 to 150 nm. They are cup-shaped under electron microscopy and contain rich mRNA, microRNA and protein components. Exosomes were first discovered in 1946. In 1987, Eberhard G. Trams and RM Johnstone first discovered a small vesicle with a membrane structure in the supernatant of sheep red blood cells cultured in vitro and named it exosomes (exos). The release mode of exosomes is different from the general budding mode. The mother cell releases granular substances to the plasma membrane, and the plasma membrane invaginates to form intraluminal vesicles. Multivesicular bodies are multivesicular endosomes containing intraluminal vesicles. Subsequently, the multivesicular bodies leave the lysosomal pathway and fuse with specific parts of the mother cell membrane to form bud vesicles. The mother cell releases the bud vesicles to the outside of the cell in the form of exocytosis. Exosomes mainly bind to receptor cells through receptor binding, enzymatic cleavage, plasma membrane fusion or endocytosis, and are taken up by receptor cells, thereby transporting the contained bioactive substances to the receptor cells to exert their regulatory effects.

[0004] Mesenchymal stem cells have low immunogenicity and immunomodulatory functions, and are safe for clinical application. Therefore, a large number of studies have reported that mesenchymal stem cells from various sources can treat skin injuries, including skin wound healing, skin incision wound healing, and skin burn treatment.

[0005] Chinese patent document CN201910948520.8 discloses a human stem cell exosome composition for anti-skin aging, comprising human adipose mesenchymal stem cell exosomes and antioxidants. The human stem cell exosome composition can block the oxidative stress aging process, reduce the intracellular reactive oxygen content of oxidatively damaged dermal fibroblasts, and improve cell proliferation ability, thereby restoring the normal thickness of the skin, enhancing the elasticity of the skin, improving skin wrinkles, reducing aging-related pigmentation, and promoting skin rejuvenation.

[0006] Most of the existing technologies are about the application and collection of exosomes, but there are few studies on promoting the production of exosomes by cells. Some use electrical stimulation to promote the secretion of exosomes by cells, and some use growth factors to promote the secretion of exosomes by cells. These may cause damage to cells or residual corresponding growth factors. Summary of the invention

[0007] In order to solve the shortcomings of the prior art, the purpose of the present invention is to provide an induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells and its application. On the one hand, the oxygen content of cell culture is reduced during the culture process, and on the other hand, functional serum-free starvation culture is adopted, which can not only avoid the introduction of exogenous substances and reduce the risk of contamination, but also promote the proliferation of mesenchymal stem cells, and effectively induce human umbilical cord mesenchymal stem cells to synthesize and secrete a large amount of exosomes.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The invention discloses an induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells, comprising a DMEM / F12 basal culture medium and an admixture, wherein the admixture comprises the following components in percentage by volume of the basal culture medium: 0.8-1.5% ITS, 0.03-0.05% oleander extract, 0.04-0.06% dandelion extract, 0.02-0.04% ginseng extract, 0.03-0.05% resveratrol, and 0.01-0.03% vitamin C.

[0010] Preferably, the additive further comprises the following components in percentage by volume of the basic culture medium: 0.03-0.05% of sheep red tannin extract, 0.01-0.03% of yam extract, and 0.01-0.02% of sodium citrate.

[0011] Preferably, the admixture includes the following components in percentage by volume of the basic culture medium: 1% ITS, 0.04% oleander extract, 0.05% dandelion extract, 0.03% ginseng extract, 0.04% resveratrol, and 0.02% vitamin C.

[0012] Preferably, the additive further comprises the following components in percentage by volume of the basal culture medium: 0.04% of sheep red tannin extract, 0.02% of yam extract, and 0.015% of sodium citrate.

[0013] Preferably, the preparation method of the oleander extract comprises the following steps: crushing the oleander under N2 flow protection, adding petroleum ether to defat, vacuum drying after crushing, adding 2 to 8 times the weight of anhydrous ethanol, constant temperature extraction at 40 to 55°C for 3 to 5 hours, decompressing and concentrating the extract, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the oleander extract with a concentration of 3 to 5 g of original medicinal material / mL;

[0014] The main active ingredients in Oleander are flavonoids, which are susceptible to oxidation. The present invention adopts a method of crushing under nitrogen protection and using petroleum ether for degreasing, which can effectively prevent the oxidation of the active ingredients and remove interfering substances; anhydrous ethanol is selected as the extraction solvent and extracted at a medium temperature (40-55°C), which can fully dissolve the flavonoids with medium polarity and avoid the damage of high temperature to the active ingredients.

[0015] Preferably, the preparation method of the dandelion extract comprises the following steps: crushing the dandelion under the protection of N2 flow, adding petroleum ether to defat, vacuum drying after crushing, adding 2 to 8 times the weight of anhydrous ethanol, and extracting at a constant temperature of 50 to 60°C for 3 to 6 hours, concentrating the extract under reduced pressure, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining a dandelion extract with a concentration of 3 to 5 g of original medicinal material / mL.

[0016] Dandelion contains active ingredients such as sesquiterpene lactones, which are also susceptible to oxidation. The nitrogen protection and degreasing pretreatment similar to that of taraxacum officinale were adopted, but the extraction temperature (50-60°C) and time (3-6h) were increased. This condition design can better destroy the plant tissue structure and increase the yield of active ingredients.

[0017] Preferably, the preparation method of the ginseng extract comprises the following steps: weighing ginseng, extracting with 60-80wt% ethanol in a water bath reflux at 70-90°C for 2-4 times, each time for 60-90 minutes, combining the extracts, concentrating under reduced pressure, recovering the ethanol until there is no alcohol taste, vacuum drying and crushing the product, dissolving it with physiological saline, filtering it through a 0.2-0.5μm filter membrane, and obtaining a ginseng extract with a concentration of 3-5g of original medicinal material / mL.

[0018] Ginsenosides are the main active ingredients of ginseng, and their extraction requires stronger conditions. The method of multiple reflux extraction with 60-80wt% ethanol at a higher temperature (70-90°C) can fully destroy the cell wall structure and dissolve saponins. Multiple extractions ensure that the active ingredients are completely extracted.

[0019] Preferably, the preparation method of the sheep red tan extract comprises the following steps: weighing sheep red tan, soaking it with 5 to 8 times its weight of deionized water, crushing and stirring it with a grinder at 40 to 50° C. for 3 to 5 hours, concentrating the suspension by pressure filtration to a relative density of 1.05 to 1.15, spray drying, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining a sheep red tan extract with a concentration of 3 to 5 g of original medicinal material / mL;

[0020] Sheep red tannins are rich in water-soluble proteins and peptides, which are sensitive to temperature. Therefore, a mild water extraction method (40-50°C) is used to ensure the extraction efficiency by appropriately extending the extraction time (3-5h). At the same time, spray drying technology is used to maintain the biological activity of active substances to the greatest extent.

[0021] Preferably, the preparation method of the yam extract comprises the following steps: weighing the yam, soaking it in 5 to 8 times its weight of deionized water, crushing and stirring it with a grinder at 40 to 60°C for 3 to 5 hours, concentrating the suspension by pressure filtration to a relative density of 1.1 to 1.2, spray drying, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the yam extract with a concentration of 3 to 5 g of original medicinal material / mL.

[0022] Yam mainly contains hydrophilic substances such as polysaccharides. The water extraction method is also used, but the temperature range is slightly wider (40-60°C), and the specifications of the extract are ensured by controlling the relative density after concentration (1.1-1.2). The spray drying process can quickly remove moisture and avoid the influence of long-term heating on the active ingredients.

[0023] The present invention also claims a use of the induction culture medium in preparing human umbilical cord mesenchymal stem cell exosomes, comprising the following steps:

[0024] (1) Isolation of human umbilical cord mesenchymal stem cells;

[0025] (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 3-5 generations of mesenchymal stem cells until the cell fusion rate reaches 70-80%, and then digesting and recovering the cells;

[0026] (3) The digested cells were inoculated into the induction medium for induction culture at a density of 1×10 5 ~5×10 5 / mL, and after hypoxic culture at 36-38°C for 48-72h, the supernatant was recovered and gradient centrifuged to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0027] Preferably, in step (3), the hypoxic culture conditions are as follows: the CO2 volume fraction is 3-8%, the O2 volume fraction is ≤1%, and the remaining gas is N2.

[0028] Preferably, in step (3), the gradient centrifugation method comprises the following steps: centrifugation at 10,000-12,000 g for 10-15 min, taking the supernatant, filtering, and centrifuging at 100,000-110,000 g for 90-100 min.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The induction culture medium provided by the present invention forms a synergistic culture environment by accurately proportioning a variety of functional admixtures. Among them, resveratrol and sodium citrate protect the structural integrity of exosomes and promote cell growth by scavenging free radicals and enhancing the activity of antioxidant enzymes; ginseng extract (containing active ingredients such as ginsenosides) and sheep red tan extract (rich in nutrients such as amino acids and peptides) can promote the proliferation of mesenchymal stem cells and inhibit their replicative aging by activating cell proliferation signaling pathways; oleander extract (containing flavonoids) and dandelion extract (containing sesquiterpene lactones, etc.) give the culture medium good antibacterial properties through their natural antibacterial active substances, effectively preventing the adverse effects of bacterial contamination on cell proliferation and exosome secretion; vitamin C, as an important antioxidant and cofactor, can promote cell metabolism and proliferation; ITS (insulin-transferrin-selenium) provides cells with essential growth factors and trace element support. This serum-free culture system not only avoids the introduction of exogenous substances, but also significantly improves the proliferation ability and paracrine effect of stem cells through the synergistic effect of each component.

[0031] 2. The preparation methods of various extracts in the present invention are optimized according to the characteristics of different raw materials: Oleander and dandelion contain easily oxidized flavonoids and polyphenols. The alcohol extraction process under nitrogen protection can effectively prevent the oxidation and inactivation of these active ingredients. At the same time, the moderate polarity of anhydrous ethanol is conducive to the extraction of these medium-polar active substances; ginsenoside components have good ethanol solubility. The ethanol reflux extraction method can fully destroy the cell wall structure under high temperature conditions and improve the extraction efficiency of saponins; sheep red tannin and yam mainly contain hydrophilic nutrients such as water-soluble proteins, polypeptides and polysaccharides. The mild water extraction method can not only maintain the biological activity of these heat-sensitive active ingredients, but also avoid the destruction of their structure by organic solvents. All extracts are filtered with a 0.2-0.5μm filter membrane. This pore size range ensures sterility and retains macromolecular active substances to the greatest extent. The entire set of extraction processes not only ensures the optimal extraction of various active ingredients, but also realizes the precise control of process parameters, laying a foundation for industrial production.

[0032] 3. The present invention innovatively optimizes the biological conditions and physicochemical properties of the culture medium during application. By reducing the oxygen content during the culture process and combining the functionalized serum-free starvation culture strategy, it not only avoids the risk of exogenous contamination, but also significantly improves the proliferation capacity of mesenchymal stem cells and the secretion of exosomes. Exosomes are collected using a standardized gradient centrifugation process to ensure the high purity and stability of the product. The entire preparation process is standardized and easy to control in quality, and has good prospects for industrial application. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the embodiments. Of course, the specific embodiments described here are only used to explain the present invention and are not used to limit the present invention.

[0034] Unless otherwise specified, the chemical reagents and materials in the present invention are purchased from commercial sources or synthesized from raw materials purchased from commercial sources.

[0035] Human umbilical cord mesenchymal stem cells were purchased from Wuhan Pronocell Life Science Co., Ltd., catalog number: CP-H165;

[0036] ITS stands for insulin-transferrin-sodium selenite culture medium supplement.

[0037] The present invention will be further described below through specific embodiments.

[0038] Preparation Example

[0039] A preparation method of a clematis extract comprises the following steps: crushing the clematis under N2 flow protection, adding petroleum ether to defat, vacuum drying after crushing, adding 8 times the weight of anhydrous ethanol, constant temperature leaching at 50°C for 4h, decompressing and concentrating the extract, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.3μm filter membrane, and obtaining the clematis extract, wherein the concentration is 5g of the original medicinal material / mL;

[0040] A preparation method of a dandelion extract comprises the following steps: crushing the dandelion under the protection of a N2 flow, adding petroleum ether to defat, vacuum drying after crushing, adding 8 times the weight of anhydrous ethanol, constant temperature leaching at 55°C for 5h, decompressing and concentrating the extract, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.3μm filter membrane, and obtaining a dandelion extract with a concentration of 5g of original medicinal material / mL;

[0041] A preparation method of ginseng extract comprises the following steps: weighing ginseng, extracting with 70wt% ethanol under water bath reflux at 80°C for 3 times, each time for 90min, combining the extracts, concentrating under reduced pressure, recovering ethanol until there is no alcohol taste, vacuum drying and crushing the product, dissolving it with physiological saline, filtering it through a 0.3μm filter membrane, and obtaining ginseng extract with a concentration of 5g raw medicinal material / mL;

[0042] A method for preparing a sheep red tan extract comprises the following steps: weighing sheep red tan, soaking it with 8 times the weight of deionized water, crushing and stirring it with a crusher at 45°C for 4 hours, filtering and concentrating the suspension to a relative density of 1.10, spray drying, dissolving it with physiological saline, filtering it with a 0.3 μm filter membrane, and obtaining a sheep red tan extract with a concentration of 5 g of original medicinal material / mL;

[0043] A method for preparing a yam extract comprises the following steps: weighing the yam, soaking it in deionized water 8 times its weight, crushing and stirring it with a crusher at 50°C for 4 hours, concentrating the suspension by pressure filtration to a relative density of 1.15, spray drying, dissolving it with physiological saline, filtering it through a 0.3 μm filter membrane, and obtaining the yam extract with a concentration of 5 g of original medicinal material / mL.

[0044] Example 1

[0045] The invention discloses an induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells, comprising a DMEM / F12 basal culture medium and an admixture, wherein the admixture comprises the following components in percentage by volume of the basal culture medium: 1.5% ITS, 0.05% oleander extract, 0.06% dandelion extract, 0.04% ginseng extract, 0.05% resveratrol, 0.03% vitamin C, 0.05% sheep red tannin extract, 0.03% yam extract, and 0.02% sodium citrate.

[0046] An application of an induction culture medium in preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0047] (1) Isolation of human umbilical cord mesenchymal stem cells;

[0048] (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 4th generation mesenchymal stem cells until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0049] (3) The digested cells were inoculated into the induction medium for induction culture at a density of 4×10 5 / mL, and the supernatant was recovered after hypoxic culture at 37°C for 60 hours. The hypoxic culture conditions were CO2 volume fraction of 4%, O2 volume fraction of 0.5%, and the remaining gas was N2. Gradient centrifugation was performed, centrifuged at 11000g for 10 min, the supernatant was taken, filtered, and centrifuged at 100000g for 100 min to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0050] Example 2

[0051] The invention discloses an induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells, comprising a DMEM / F12 basal culture medium and an admixture, wherein the admixture comprises the following components in percentage by volume of the basal culture medium: 1.2% ITS, 0.04% oleander extract, 0.05% dandelion extract, 0.03% ginseng extract, 0.04% resveratrol, 0.02% vitamin C, 0.04% sheep red tannin extract, 0.02% yam extract, and 0.015% sodium citrate.

[0052] An application of an induction culture medium in preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0053] (1) Isolation of human umbilical cord mesenchymal stem cells;

[0054] (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 4th generation mesenchymal stem cells until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0055] (3) The digested cells were inoculated into the induction medium for induction culture at a density of 4×10 5 / mL, and the supernatant was recovered after hypoxic culture at 37°C for 60 hours. The hypoxic culture conditions were CO2 volume fraction of 4%, O2 volume fraction of 0.5%, and the remaining gas was N2. Gradient centrifugation was performed, centrifuged at 11000g for 10 min, the supernatant was taken, filtered, and centrifuged at 100000g for 100 min to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0056] Example 3

[0057] The invention discloses an induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells, comprising a DMEM / F12 basal culture medium and an admixture, wherein the admixture comprises the following components in percentage by volume of the basal culture medium: 0.8% ITS, 0.03% oleander extract, 0.04% dandelion extract, 0.02% ginseng extract, 0.03% resveratrol, 0.01% vitamin C, 0.03% sheep red tannin extract, 0.01% yam extract, and 0.01% sodium citrate.

[0058] An application of an induction culture medium in preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0059] (1) Isolation of human umbilical cord mesenchymal stem cells;

[0060] (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 4th generation mesenchymal stem cells until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0061] (3) The digested cells were inoculated into the induction medium for induction culture at a density of 4×10 5 / mL, and the supernatant was recovered after hypoxic culture at 37°C for 60 hours. The hypoxic culture conditions were CO2 volume fraction of 4%, O2 volume fraction of 0.5%, and the remaining gas was N2. Gradient centrifugation was performed, centrifuged at 11000g for 10 min, the supernatant was taken, filtered, and centrifuged at 100000g for 100 min to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0062] Comparative Example 1

[0063] A method for preparing human umbilical cord mesenchymal stem cell exosomes comprises the following steps:

[0064] (1) Isolation of human umbilical cord mesenchymal stem cells;

[0065] (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 4th generation mesenchymal stem cells until the cell fusion rate reaches 75%, and then digesting and recovering the cells;

[0066] (3) The digested cells were inoculated in DMEM / F12 medium (supplemented with 1.5% ITS) at a density of 4×10 5 / mL, and the supernatant was recovered after hypoxic culture at 37°C for 60 hours. The hypoxic culture conditions were CO2 volume fraction of 4%, O2 volume fraction of 0.5%, and the remaining gas was N2. Gradient centrifugation was performed, centrifuged at 11000g for 10 min, the supernatant was taken, filtered, and centrifuged at 100000g for 100 min to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

[0067] Under sterile conditions, umbilical cord mesenchymal stem cells cultured to the fourth generation in step (2) of Examples 1 to 3 and Comparative Example 1 were collected. The cells were suspended in pre-cooled PBS containing 10% FCS and 1% sodium azide, and the cell concentration was adjusted to 3×10 6 Add 100 μL of cell suspension (3×10 5 Cells) and corresponding fluorescent labeled antibodies were incubated at 4°C in the dark for 30 min. The cells were washed three times by centrifugation using precooled PBS containing 10% FCS and 1% sodium azide, and detected using flow cytometry. The test results are shown in Table 1.

[0068] Table 1 Flow cytometry detection results of exosome markers (%)

[0069]

[0070]

[0071] Determination of exosome protein concentration: BCA protein quantification kit and ND-2000 ultra-micro UV-visible spectrophotometer were used to detect the total amount of exosome protein. Parameters: 0.1 mL of sample + 2.0 mL of reagent; reaction time 30 min; reaction temperature 37 ° C; wavelength 562 nm. The supernatants obtained from the cultures of Examples 1 to 3 and Comparative Example 1 were separated to obtain 50 mL of exosome suspension, and the protein concentrations were shown in Table 2.

[0072] Table 2 Exosome protein content in Examples and Comparative Examples

[0073]

[0074] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. An induction culture medium for preparing exosomes of human umbilical cord mesenchymal stem cells, characterized in that: The invention comprises a DMEM / F12 basic culture medium and an additive, wherein the additive comprises the following components in percentage by volume of the basic culture medium: 0.8-1.5% ITS, 0.03-0.05% oleander extract, 0.04-0.06% dandelion extract, 0.02-0.04% ginseng extract, 0.03-0.05% resveratrol and 0.01-0.03% vitamin C.

2. The induction medium according to claim 1, characterized in that The additive also comprises the following components in percentage by volume of the basic culture medium: 0.03-0.05% of sheep red tannin extract, 0.01-0.03% of yam extract and 0.01-0.02% of sodium citrate.

3. The induction medium according to claim 1, characterized in that The additive comprises the following components in percentage by volume of the basic culture medium: 1% ITS, 0.04% oleander extract, 0.05% dandelion extract, 0.03% ginseng extract, 0.04% resveratrol, and 0.02% vitamin C.

4. The induction medium according to claim 1, characterized in that The additive also includes the following components in percentage by volume of the basic culture medium: 0.04% of sheep red tannin extract, 0.02% of yam extract, and 0.015% of sodium citrate.

5. The induction medium according to claim 1, characterized in that The preparation method of the oleander extract comprises the following steps: crushing the oleander under the protection of N2 flow, adding petroleum ether to defat, vacuum drying after crushing, adding 2 to 8 times the weight of anhydrous ethanol, constant temperature leaching at 40 to 55° C. for 3 to 5 hours, decompressing and concentrating the extract, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the oleander extract, wherein the concentration is 3 to 5 g of the original medicinal material / mL; The preparation method of the dandelion extract comprises the following steps: crushing the dandelion under the protection of N2 flow, adding petroleum ether for degreasing, vacuum drying after crushing, adding 2 to 8 times the weight of anhydrous ethanol, constant temperature leaching at 50 to 60°C for 3 to 6 hours, decompressing and concentrating the extract, evaporating and crushing the product at low temperature, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the dandelion extract with a concentration of 3 to 5 g of original medicinal material / mL.

6. The induction medium according to claim 1, characterized in that The preparation method of the ginseng extract comprises the following steps: weighing ginseng, extracting 2 to 4 times with 60 to 80 wt% ethanol at 70 to 90° C. in a water bath under reflux, each time for 60 to 90 minutes, combining the extracts, concentrating under reduced pressure, recovering the ethanol until there is no alcohol taste, vacuum drying and crushing the product, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the ginseng extract with a concentration of 3 to 5 g of original medicinal material / mL.

7. The induction medium according to claim 2 or 4, characterized in that The preparation method of the sheep red tan extract comprises the following steps: weighing the sheep red tan, soaking it with 5-8 times the weight of deionized water, crushing and stirring it with a crusher at 40-50°C for 3-5 hours, filtering and concentrating the suspension to a relative density of 1.05-1.15, spray drying, dissolving it with physiological saline, filtering it with a 0.2-0.5 μm filter membrane, and obtaining the sheep red tan extract with a concentration of 3-5 g of original medicinal material / mL; The preparation method of the yam extract comprises the following steps: weighing the yam, soaking it in deionized water 5 to 8 times its weight, crushing and stirring it with a crusher at 40 to 60° C. for 3 to 5 hours, filtering and concentrating the suspension to a relative density of 1.1 to 1.2, spray drying, dissolving it with physiological saline, filtering it through a 0.2 to 0.5 μm filter membrane, and obtaining the yam extract with a concentration of 3 to 5 g of original medicinal material / mL.

8. Use of the induction medium according to any one of claims 1 to 7 in preparing exosomes of human umbilical cord mesenchymal stem cells, characterized in that: The steps include: (1) Isolation of human umbilical cord mesenchymal stem cells; (2) subculturing human umbilical cord mesenchymal stem cells in DMEM / F12 medium, culturing 3-5 generations of mesenchymal stem cells until the cell fusion rate reaches 70-80%, and then digesting and recovering the cells; (3) The digested cells were inoculated into the induction medium for induction culture at a density of 1×10 5 ~5×10 5 / mL, and after hypoxic culture at 36-38°C for 48-72h, the supernatant was recovered and gradient centrifuged to collect exosomes to obtain human umbilical cord mesenchymal stem cell exosomes.

9. The use according to claim 8, characterized in that: In step (3), the hypoxic culture conditions are as follows: the CO2 volume fraction is 3-8%, the O2 volume fraction is ≤1%, and the remaining gas is N2.

10. The use according to claim 8, characterized in that: In step (3), the gradient centrifugation method comprises the following steps: centrifugation at 10,000 to 12,000 g for 10 to 15 min, taking the supernatant, filtering, and centrifuging at 100,000 to 110,000 g for 90 to 100 min.

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