An induction culture medium, method and application for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment

Abstract

The embodiment of the present invention discloses an induction culture medium, method and application for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The induction culture medium includes a basal culture medium and an induction drug, and the induction drug includes transforming growth factor β, hepatocyte growth factor, cyclic adenosine monophosphate, triptolide, sodium hydrosulfide, lipopolysaccharide, hyaluronic acid, dexamethasone, L-glutamine and HEPES buffer. The induction culture medium provided by the present invention can promote the metabolism and energy production of hair follicle-derived mesenchymal stem cells, enhance the antioxidant capacity of cells, improve the survival rate and factor secretion ability of cells in a hypoxic environment, and solve the problems of low cell viability, slow growth, low cytokine secretion, etc. after transplanting cells into damaged tissue areas in the prior art.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedicine technology, and specifically to an induction culture medium, method and application for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment. Background Art

[0002] At present, the main means of treating hair loss include drug therapy, hair follicle transplantation and other auxiliary treatments. Drug treatments such as minoxidil are effective in promoting hair growth, but they need to be used for a long time and the effect may disappear after stopping the drug. Finasteride is suitable for male pattern baldness and can slow down the process of hair loss, but it may cause some side effects, such as sexual dysfunction. Hair follicle transplantation is to transplant healthy hair follicles from the donor area of ​​the head to the hair loss area, which can restore hair growth, but the cost of the operation is high. Other auxiliary treatments such as laser treatment and traditional Chinese medicine treatment have problems such as limited treatment effect and long treatment cycle.

[0003] Cell transplantation for hair loss is an emerging treatment method. Cell transplantation to promote hair growth after scalp is a complex process involving cell survival, proliferation and the final hair growth effect. After cell transplantation, the problems faced by cells, such as hypoxia, ischemia and nutrient deficiency, are key factors affecting cell function and the final effect. After cell transplantation to the scalp, cells may encounter insufficient local blood flow or insufficient angiogenesis, causing the cells to be in a state of hypoxia. Hypoxia can inhibit the normal function of cells, including cell proliferation, differentiation and survival. In a hypoxic environment, cells may activate the regulatory mechanism of hypoxia-inducible factor (HIF) to adapt to the low oxygen environment, but excessive hypoxic response may lead to cell apoptosis or dysfunction. Ischemia refers to a reduction in blood supply to local tissues or organs, which may result in insufficient oxygen and nutrients for cells in the cell transplantation area. Ischemia can cause cell metabolic disorders, affecting cell survival and function. Long-term ischemia may also cause tissue necrosis and affect the effect of cell transplantation. After cell transplantation, cells may not be able to obtain sufficient nutrients from surrounding tissues, including growth factors, amino acids, carbohydrates, etc. Nutritional deficiency directly affects cell proliferation, differentiation and function, which may lead to slow cell growth, abnormal differentiation or impaired function. Lack of nutrients may also aggravate the oxidative stress state of cells and further damage cells. Therefore, the promotion of scalp hair growth after cell transplantation requires comprehensive consideration of the problems of cell hypoxia, ischemia and nutritional deficiency, and the adoption of corresponding strategies for targeted solutions to improve cell survival rate and hair growth effect. Summary of the invention

[0004] To this end, the embodiments of the present invention provide an induction culture medium, method and application for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment, so as to solve the problems of low cell viability, slow growth and low cytokine secretion levels after transplanting cells into damaged tissue areas in the prior art.

[0005] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] According to a first aspect of an embodiment of the present invention, the present invention provides an induction medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment, the induction medium comprising a basal medium and an induction drug, the induction drug comprising transforming growth factor β 15-25 ng / ml, hepatocyte growth factor 8-15 ng / ml, cyclic adenosine monophosphate 1-2.5 mM, triptolide 0.03-0.05 μM, sodium hydrosulfide 220-280 μM, lipopolysaccharide 0.4-0.6 g / ml, hyaluronic acid 5-15 μg / ml, dexamethasone 0.8-0.12 μM, L-glutamine 40-60 ng / ml, and HEPES buffer 15-25 mM.

[0007] The effects of the various components in the induction drug of the present invention are as follows:

[0008] Transforming growth factor β TGF-β (P1230) is an important cytokine that plays an important role in biological processes such as cell growth, differentiation, apoptosis, and extracellular matrix synthesis.

[0009] Hepatocyte growth factor HGF (HY-P7121) has multiple biological functions, including promoting the growth and proliferation of various cell types, enhancing cell migration and invasion ability, and promoting cell differentiation, especially in hepatocyte differentiation; at the same time, it is also involved in cell protection and repair processes, such as promoting wound healing and organ regeneration, and has anti-inflammatory and immunomodulatory effects, regulating inflammation and immune responses by inhibiting inflammatory cell activation and promoting immune cell function.

[0010] Cyclic adenosine monophosphate (CAMP) (CB8747868) is an important substance involved in regulating metabolism and biological functions in cells, and is the "second messenger" of life information transmission. In the body, it can promote the survival of myocardial cells, enhance the ability of myocardial cells to resist damage, ischemia and hypoxia; promote the flow of calcium ions into myocardial cells, enhance phosphorylation, promote excitation-contraction coupling, improve myocardial cell contractility, and increase cardiac output; at the same time, it also dilates peripheral blood vessels, reduces cardiac ejection impedance, reduces cardiac preload and postload, increases cardiac output, and improves cardiac function. It thus plays a role in nourishing the myocardium, exerting positive inotropic force, vasodilation, anti-platelet aggregation, and anti-arrhythmia.

[0011] Triptolide (CAS No.: 38748-32-2) is a diterpene lactone compound extracted from the root of Tripterygium wilfordii, a traditional Chinese medicine, which has significant immunosuppressive, anti-inflammatory and anti-tumor activities. More and more studies have shown that triptolide has a significant effect in the treatment of inflammatory and autoimmune diseases including rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis and nephritis. The anti-inflammatory mechanism of Tripterygium wilfordii is mainly through regulating the activity of key proteins in the inflammatory signaling pathways such as MAPK, PI3K, Jak2, Bc1-2 / Bax8, JNK-1 / 2, MPK1, ERK-1 / 2, NF-KB, active ROS and caspase 8, thereby inhibiting the production of NO, COX-2 expression and down-regulating the expression levels of inflammatory cytokines such as TNF-α, IL-1β and IL-6.

[0012] Sodium hydrosulfide (NaHS) (CAS No. 1310-73-2) has a certain antioxidant effect. Through its antioxidant properties, it can help reduce the free radicals accumulated in the cell culture environment, thereby protecting cells from damage by oxidative stress.

[0013] Lipopolysaccharide (LPS297-473-0) has a variety of biological activities, including immune activation, inflammation induction and cytoprotection. In pluripotent stem cells (MSCs), the role of LPS is mainly reflected in two aspects: protecting MSCs from apoptosis induced by oxidative stress and promoting the proliferation of MSCs.

[0014] Hyaluronic acid (CAS No.: 9004-61-9) Hyaluronic acid can regulate the microenvironment of stem cells, thereby affecting the biological behavior of stem cells, including key processes such as proliferation, differentiation and migration. Studies have shown that hyaluronic acid provides necessary nutrition and growth factors for stem cells by providing a suitable extracellular matrix, thereby promoting their proliferation. In addition, the molecular weight and concentration of hyaluronic acid can also affect the differentiation direction of stem cells. High molecular weight hyaluronic acid may promote the differentiation of stem cells into cartilage or nerve cells, while low molecular weight hyaluronic acid may promote the differentiation of stem cells into adipocytes or osteoblasts.

[0015] Dexamethasone (CAS No.: 50-02-2), as a glucocorticoid, plays an important role in stem cell research. Its main functions include promoting stem cell proliferation, regulating stem cell differentiation, inhibiting immune response, and promoting angiogenesis. Through these effects, dexamethasone has a multi-faceted impact on stem cell research: First, dexamethasone can stimulate stem cells to enter the cell cycle, accelerate their proliferation process, and provide sufficient cell resources for stem cell research. Secondly, it exhibits the ability to regulate gene expression during stem cell differentiation and can affect the differentiation direction of stem cells into different cell types, which is particularly important in the generation of induced pluripotent stem cells (iPSCs). Dexamethasone works together with other inducers (such as YAP, SOX2, etc.) to promote the transformation of somatic cells such as fibroblasts into pluripotent stem cells.

[0016] L-glutamine (CAS No.: 56-85-9) is an important raw material for cells to synthesize nucleic acids and proteins. In cell culture, glutamine deficiency can lead to poor cell growth or even death.

[0017] HEPES buffer (CAS No. 7365-45-9) helps maintain a stable pH value in cell culture medium, which is essential for the growth and function of stem cells. Cells need a relatively constant pH environment to carry out normal metabolic activities.

[0018] Furthermore, the inducing drugs include transforming growth factor β 20 ng / ml, hepatocyte growth factor 10 ng / ml, cyclic adenosine monophosphate 2 mM, triptolide 0.04 μM, sodium hydrosulfide 250 μM, lipopolysaccharide 0.5 g / ml, hyaluronic acid 10 μg / ml, dexamethasone 0.1 μM, L-glutamine 50 ng / ml, and HEPES buffer 20 mM.

[0019] Furthermore, the basic culture medium is DMEM / F12.

[0020] According to a second aspect of an embodiment of the present invention, the present invention provides a method for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment, wherein the hair follicle-derived mesenchymal stem cells are cultured using the induction culture medium as described in any one of the above items.

[0021] Furthermore, the method includes: inoculating and culturing the 3rd to 5th generation hair follicle-derived mesenchymal stem cells, replacing the induction culture medium with the culture medium after the cells are completely attached to the wall, and culturing in a three-gas incubator at 37° C., 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0022] Furthermore, the inoculation culture conditions are: 37° C., 5% CO 2 and 95% air, with DMEM / F-12 containing 10 v / v% fetal bovine serum as the culture medium.

[0023] According to a third aspect of an embodiment of the present invention, the present invention provides use of the induction culture medium as described in any one of the above items in preparing a product for preventing and treating hair loss.

[0024] According to a fourth aspect of the embodiments of the present invention, the present invention provides use of the method as described in any one of the above items in preparing a product for preventing and treating hair loss.

[0025] The embodiments of the present invention have the following advantages:

[0026] The induction culture medium provided by the present invention can promote the metabolism and energy production of hair follicle-derived mesenchymal stem cells, enhance the antioxidant capacity of cells, and improve the survival rate and factor secretion capacity of cells in a hypoxic environment, thereby solving the problems of low cell viability, slow growth, and low cytokine secretion levels after cells are transplanted into damaged tissue areas in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0028] Figure 1 The test results of cell proliferation in each group provided by the present invention, wherein AC is the experimental group 1-3, DF is the control group 1-3, and G is the blank control group;

[0029] Figure 2 The cell number detection results of each group provided by the present invention;

[0030] Figure 3 The cell proliferation activity test results of each group provided by the present invention;

[0031] Figure 4 These are the test results of the cytokine secretion capacity of each group provided by the present invention. DETAILED DESCRIPTION

[0032] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0034] Example 1

[0035] The present embodiment provides an induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The raw materials are DMEM / F12 and induction drugs. The composition of the induction drugs is as follows: transforming growth factor β 20 ng / ml, hepatocyte growth factor 10 ng / ml, cyclic adenosine monophosphate 2 mM, triptolide 0.04 μM, sodium hydrosulfide 250 μM, lipopolysaccharide 0.5 g / ml, hyaluronic acid 10 μg / ml, dexamethasone 0.1 μM, L-glutamine 50 ng / ml, and HEPES buffer 20 mM.

[0036] Example 2

[0037] The present embodiment provides an induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The raw materials are DMEM / F12 and induction drugs. The composition of the induction drugs is as follows: transforming growth factor β 15 ng / ml, hepatocyte growth factor 8 ng / ml, cyclic adenosine monophosphate 1 mM, triptolide 0.05 μM, sodium hydrosulfide 225 μM, lipopolysaccharide 0.4 g / ml, hyaluronic acid 6 μg / ml, dexamethasone 0.08 μM, L-glutamine 40 ng / ml, and HEPES buffer 15 mM.

[0038] Example 3

[0039] The present embodiment provides an induction medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The raw materials are DMEM / F12 and induction drugs. The composition of the induction drugs is as follows: transforming growth factor β 25 ng / ml, hepatocyte growth factor 12 ng / ml, cyclic adenosine monophosphate 2.3 mM, triptolide 0.03 μM, sodium hydrosulfide 280 μM, lipopolysaccharide 0.58 g / ml, hyaluronic acid 14 μg / ml, dexamethasone 0.12 μM, L-glutamine 60 ng / ml, and HEPES buffer 24 mM.

[0040] Comparative Example 1

[0041] The comparative example provides an induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The raw materials are DMEM / F12 and induction drugs. The composition of the induction drugs is: epidermal growth factor 20 ng / ml, hepatocyte growth factor 10 ng / ml, cyclic adenosine monophosphate 2 mM, triptolide 0.04 μM, sodium hydrosulfide 250 μM, lipopolysaccharide 0.5 g / ml, hyaluronic acid 10 μg / ml, dexamethasone 0.1 μM, L-glutamine 50 ng / ml, and HEPES buffer 20 mM.

[0042] Comparative Example 2

[0043] This comparative example provides an induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The difference between this comparative example and Example 1 is that the basic culture medium used is DMEM.

[0044] Comparative Example 3

[0045] The comparative example provides an induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment. The raw materials are DMEM / F12 and induction drugs. The composition of the induction drugs is: transforming growth factor β 35 ng / ml, hepatocyte growth factor 10 ng / ml, cyclic adenosine monophosphate 2 mM, triptolide 0.04 μM, sodium hydrosulfide 150 μM, lipopolysaccharide 0.5 g / ml, hyaluronic acid 10 μg / ml, dexamethasone 0.1 μM, L-glutamine 80 ng / ml, and HEPES buffer 20 mM.

[0046] Example 4

[0047] This embodiment provides a method for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment, comprising:

[0048] (1) Obtaining hair follicle-derived mesenchymal stem cells

[0049] Materials: 1 sterile operating room, 500ml of iodine disinfectant, 1 bottle of 75% medical alcohol, 2 tubes of lidocaine, 2 packs of sterile gauze, 2 packs of sterile cotton balls, a hair follicle extractor, 2 hair follicle extractor heads, 1 bottle of PBS buffer, 1 bottle of DMEM / F12 culture medium, serum, growth factors, and 2 6-well plates.

[0050] In vitro culture: The extracted hair follicle tissue was cut into small pieces, and DMEM / F-12 culture medium (containing 10v / v% fetal bovine serum) was evenly added to the well plate, and cultured in a cell culture incubator at 37°C, 5% CO2 and 95% air. After 48 hours, half of the medium was replaced, and the migration of stem cells from the tissue blocks was observed after 7-10 days.

[0051] Passaging: When cells reach approximately 80% confluence, they are ready for passaging.

[0052] Digestion of cells: Aspirate the culture medium in the culture flask and wash the cells once with PBS. Then add 0.25% trypsin and incubate at 37°C for about 3 minutes. Observe under an inverted microscope. When the cells shrink and become round, aspirate the digestion solution and add DMEM / F-12 culture medium (containing 10v / v% fetal bovine serum) to terminate digestion.

[0053] Blowing and inoculation: Use a pipette to gently blow the cells to make them fall off the wall of the culture bottle and disperse into a single cell suspension. After counting, inoculate and subculture. Then add fresh complete culture medium and continue to culture in a cell culture incubator at 37°C, 5% CO2 and 95% air.

[0054] Culture observation: After the cells are completely attached to the wall, culture observation is performed. After 48 hours, fresh complete culture medium is replaced to maintain a good growth environment for the cells.

[0055] Identification: Morphological characteristics are used for identification. Under a light microscope, hair follicle-derived mesenchymal stem cells usually appear as cells that grow on the wall and have a long spindle-shaped morphology.

[0056] (2) The third generation hair follicle-derived mesenchymal stem cells were seeded on well plates of different areas and placed in DMEM / F-12 culture medium (containing 10 v / v% fetal bovine serum). They were cultured in an incubator at 37°C, 5% CO2 and 95% air for 4 h to allow them to completely adhere to the wall. The supernatant of the culture medium in the culture dish was then aspirated and the cells were washed three times with PBS buffer. After the residual PBS buffer was aspirated, the induction medium was slowly added along the wall of the well plate and the cells were cultured in a three-gas incubator at 37°C, 1% O2, 5% CO2 and 94% N2.

[0057] Test Example 1

[0058] According to the method of Example 4, the third generation hair follicle-derived mesenchymal stem cells were inoculated on a 24-well plate with an inoculation amount of 0.5×10^5, and DMEM / F-12 (containing 10 v / v% fetal bovine serum) was used as the culture medium. They were cultured in an incubator at 37° C., 5% CO2 and 95% air for 4 hours. After they were completely attached to the wall, the induction culture medium of Examples 1-3 and Comparative Examples 1-3 was used to culture them in a three-gas incubator at 37° C., 1% O2, 5% CO2, and 94% N2 (respectively set as experimental groups 1-3 and control groups 1-3), and a blank control group was set at the same time.

[0059] The cell culture method of the blank control group was as follows: after the cells were cultured according to the above method and completed adhesion, they were cultured in a three-gas incubator at 37°C, 1% O2, 5% CO2, and 94% N2 using DMEM / F-12 medium.

[0060] 1. Observe the proliferation status of cells using an inverted microscope

[0061] On the third day of culture, the proliferation status of cells in each group was observed under an inverted microscope. Figure 1 The cell proliferation conditions of the experimental groups 1-3, the control groups 1-3 and the blank control group were different, specifically manifested in the differences in cell number, morphology and arrangement. On the third day, the cells cultured in the blank control group had a significant increase in cell volume and irregular cell outlines. The cell morphology of the control groups 1-3 showed irregular characteristics, accompanied by the appearance of a large number of dead cells. The cell volume of the experimental group 1-3 was significantly smaller than that of the control group 1-3, and the cell outline was clearer, with a greater number of live cells.

[0062] 2. Cell number detection

[0063] At 12h, 1d, 3d, 5d, and 7d of culture, the number of cells in each group was measured using a cell counter. The results are shown in Table 1 below.

[0064] Table 1

[0065]

[0066] The trend of cell number changes can be seen in Figure 2 We observed that within the time range of 12 hours to 1 day, the number of cells in experimental groups 1-3, control groups 1-3, and the blank control group all showed an increasing trend. However, after 1 day, the number of cells in the blank control group first began to decline, and on the 7th day it dropped below the inoculation amount, indicating that the cells in this group experienced a large number of cell deaths. The number of cells in experimental groups 1-3 and control group 1 increased from 1 to 5 days, but the growth rate was significantly lower than that of experimental group 1. The number of cells in control groups 2-3 began to decrease after the 3rd day, while the number of cells in experimental groups 1-3 began to decrease after the 5th day. By the 7th day, the number of cells in experimental group 1 was still significantly higher than that in other groups.

[0067] 3. CCK-8 method for cell proliferation activity detection

[0068] The cell inoculation volume was adjusted to 1000 cells / well, inoculated in 96-well plates, and replicated in 5 wells. Grouping and culture were performed in the same manner as above. 100 μL / well CCK-8 solution (the volume ratio of complete culture medium to CCK-8 was 9:1) was added at 1, 3, 5, and 7 days, respectively. The absorbance value at a wavelength of 450 nm was measured by a microplate reader for 2.5 hours of culture. The results are shown in Table 2 below.

[0069] Table 2

[0070]

[0071] Results Figure 3The OD values ​​of experimental group 1 were higher than those of other groups at 1, 3, 5, and 7 days, and were most obvious at 3 days, which proved that the cell activity of experimental group 1 decreased with the increase of hypoxia time, but it was not particularly serious. In the blank control group and control groups 1-3, the cell proliferation activity was weaker with the increase of hypoxia time, and it decreased significantly with the increase of hypoxia time. In experimental groups 2 and 3, the cell proliferation activity increased slowly with the increase of hypoxia time, and began to decrease after the third day. The results presented were also better than those of control groups 1-3.

[0072] 4. In vitro detection of cytokine secretion ability

[0073] The cell inoculation volume was adjusted to 1×10^6 and inoculated on a 6-well plate. The cells were grouped and cultured in the same manner as above. After culturing for 3 days in a three-gas incubator at 37°C, 1% O2, 5% CO2, and 94% N2, the supernatant of each group was analyzed using RayBiotech's L antibody array technology. A total of 9 key proteins were detected in this experiment, including interleukin-15 (IL-15), interleukin-18 (IL-18), basic fibroblast growth factor (FGF Basic), leukemia inhibitory factor (LIF), macrophage colony-stimulating factor (M-CSF), monocyte chemoattractant protein (MIG), macrophage inflammatory protein 2 (MIP2), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor (VEGF). Each antibody on the chip was repeated twice. In data analysis, the background-removed fluorescence signal FI (F532 Median-B532 Median) was used for analysis, and the mean of two replicates of all antibodies was calculated. The mean was used as the signal value of each antibody for subsequent analysis. In the chip experiment results, for proteins with low signal values, signal values ​​FI < 25 were considered undetected. The detection results of each cytokine are shown in Tables 3 and Figure 4 .

[0074] Table 3

[0075]

[0076] The results showed that among the 9 proteins detected in experimental group 1, the concentrations of IL-15, basic fibroblast growth factor (FGFBasic), leukemia inhibitory factor (LIF), macrophage colony stimulating factor (M-CSF) and vascular endothelial growth factor (VEGF) were significantly different from those in other groups. Specifically:

[0077] The concentration of VEGF in experimental group 1 was significantly higher than that in other groups, about 4-5 times that of the blank control group and experimental group 2, and 8 times that of experimental group 3. As a potent angiogenic factor, VEGF can promote the proliferation and migration of scalp vascular endothelial cells and plays a key role in the growth and maintenance of hair. The concentration of M-CSF in experimental group 1 was significantly higher than that in the blank control group and experimental group 3 (about 6 times), and experimental group 2 (about 9-10 times). M-CSF plays an important role in the immune system and hematopoietic cells, and its specific role in the hair growth process may be closely related to the maintenance of the scalp microenvironment. The concentration of LIF was significantly increased in experimental group 1, about 17 times that of the blank control group, 23 times that of experimental group 2, and 10 times that of experimental group 3. As a multifunctional cytokine, LIF can not only promote the self-renewal and differentiation of stem cells, but also participate in the regulation of immune response and inflammatory processes. The concentration of FGF Basic in experimental group 1 was significantly higher than that in the blank control group and experimental group 2 (about 13-14 times), and experimental group 3 (about 3 times). FGF Basic is involved in regulating biological processes such as cell proliferation, differentiation and migration, and has a promoting effect on scalp angiogenesis and hair follicle cell proliferation. The concentration of IL-15 in experimental group 1 was significantly higher than that in the blank control group and experimental group 2 (about 2-3 times), and experimental group 3 (about 8 times). As a cytokine secreted by interstitial cells, IL-15 plays a role in promoting the proliferation, differentiation and activation of immune cells, and also plays an important role in regulating immune responses and antiviral effects. It is worth noting that the concentrations of IL-18, monocyte chemoattractant protein (MIG), macrophage inflammatory protein 2 (MIP 2), and platelet-derived growth factor BB (PDGF-BB) did not differ significantly among the groups. However, the concentrations of these proteins in experimental group 1 were generally higher than those in other groups. This finding suggests that although the concentrations of some proteins did not differ significantly among the groups, experimental group 1 may have specific biological effects that deserve further study (such as Figure 4 ).

[0078] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. An induction culture medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment, characterized in that: The induction culture medium comprises a basal culture medium and an induction drug, wherein the induction drug comprises transforming growth factor β 15-25 ng / ml, hepatocyte growth factor 8-15 ng / ml, cyclic adenosine monophosphate 1-2.5 mM, triptolide 0.03-0.05 μM, sodium hydrosulfide 220-280 μM, lipopolysaccharide 0.4-0.6 g / ml, hyaluronic acid 5-15 μg / ml, dexamethasone 0.08-0.12 μM, L-glutamine 40-60 ng / ml, and HEPES buffer 15-25 mM.

2. The induction medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment according to claim 1, characterized in that: The inducing drugs include transforming growth factor β 20 ng / ml, hepatocyte growth factor 10 ng / ml, cyclic adenosine monophosphate 2 mM, triptolide 0.04 μM, sodium hydrosulfide 250 μM, lipopolysaccharide 0.5 g / ml, hyaluronic acid 10 μg / ml, dexamethasone 0.1 μM, L-glutamine 50 ng / ml, and HEPES buffer 20 mM.

3. The induction medium for improving the viability and factor secretion ability of hair follicle-derived mesenchymal stem cells in a hypoxic environment according to claim 1, characterized in that: The basic culture medium is DMEM / F12.

4. A method for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment, characterized in that: The hair follicle-derived mesenchymal stem cells are cultured using the induction medium described in any one of claims 1 to 3.

5. The method for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment according to claim 4, characterized in that: The method comprises: The 3rd to 5th generation hair follicle-derived mesenchymal stem cells were inoculated and cultured. After the cells were completely attached to the wall, the induction medium was replaced and cultured in a three-gas incubator at 37° C., 1% O 2 , 5% CO 2 , and 94% N 2 for 72 hours.

6. The method for improving the viability and factor secretion capacity of hair follicle-derived mesenchymal stem cells in a hypoxic environment according to claim 5, characterized in that: The inoculation culture conditions are: 37° C., 5% CO 2 and 95% air, with DMEM / F-12 containing 10 v / v% fetal bovine serum as the culture medium.

7. Use of the induction culture medium according to any one of claims 1 to 3 in preparing a product for preventing and treating hair loss.

8. Use of the method according to any one of claims 4 to 6 in preparing a product for preventing and treating hair loss.

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