Mesenchymal stem cell hair growth liquid and preparation method thereof

Abstract

The invention relates to a mesenchymal stem cell hair growth liquid and a preparation method thereof. The hair growth liquid comprises mesenchymal stem cells, the mesenchymal stem cells carry Wnt-1a protein and vasoactive factors, and the vasoactive factors comprise an endothelial cell growth factor (VEGF) and angiopoietin Ang-1. According to the invention, mesenchymal stem cells are stimulated by using a traditional Chinese medicine monomer combined with a hypoxia pretreatment method, expression and secretion of Wnt-1a protein, vascular endothelial cell growth factor (VEGF) and angiogenin 1 (Ang-1) in the product are greatly improved, and the obtained hair growth liquid product can effectively promote human hair regeneration through an intradermal introduction method, so that the hair growth effect is improved. The traditional Chinese medicine composition can be used for preventing and treating alopecia caused by exuberant secretion of male hormone.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a mesenchymal stem cell hair growth fluid and a preparation method thereof. Background Art

[0002] Excessive stress in modern society, improper diet, poor rest, tension, anxiety, depression, excessive workload, etc. will immediately affect the endocrine system, leading to disorders in the metabolism of female and male growth hormones, causing excessive secretion of male hormones, and leading to hair loss. In addition to traditional Chinese medicine treatment, there is also Western medicine treatment, but the effects of these two drugs are often not completely satisfactory; Chinese medicine preparations for hair loss often have unclear effects, or some Chinese medicines have serious toxic side effects, and Western medicines mostly use hormone treatment, and the effect is prone to repeated changes.

[0003] The hair follicle is a tiny but delicate organ that has its own unique formation method and physiological cycle. Healthy hair follicles will go through three stages: growth phase, regression phase, and resting phase, and cycle back and forth. In the growth phase, epithelial stem cells grow toward the subcutaneous tissue through intercellular signaling and cell differentiation to form the hair follicle structure; in the regression phase, the hair stops growing, and the lowest hair papilla retracts upward to a special part of the hair follicle structure - the bulge, until the next cell signal stimulates the hair follicle stem cells to grow again. The process of hair follicles re-entering the growth phase from the resting phase is called hair follicle regeneration, and the hair follicle stem cells located in the bulge are responsible for hair follicle regeneration.

[0004] Dermal papilla (DP) cells are the main components of the dermis and are considered to be the main signaling center responsible for maintaining hair growth and controlling the hair follicle cycle. The microenvironment can affect DP cells to promote hair growth, and a bad microenvironment can lead to hair loss. Most human males with androgenic alopecia have intact hair follicle stem cells, mainly due to the loss of the DP signaling pathway, which prevents the start of the hair follicle growth phase. Therefore, restoring the ability of DP cells to induce hair growth is crucial for the treatment of hair loss.

[0005] Mesenchymal stem cells (MSC) have been widely used in regenerative medicine, and more and more in vitro and in vivo studies have shown that the paracrine function of MSC plays an important role. The pleiotropic growth factors secreted by MSC during the regeneration and repair process can assist in activating the hair-inducing ability of DP cells. MSC can secrete a variety of active factors, and changing some culture conditions and adding certain small molecule compounds during in vitro culture can effectively promote the paracrine function of MSC. For example, adding vitamins A, B, C, and D in CN101400787A can effectively promote the secretion function of adipose mesenchymal stem cells (ADSC); in CN201710015650.7, adding small molecule compounds such as coenzyme A, cocarboxylase, and flavin adenine dinucleotide combined with hypoxic culture can promote adipose mesenchymal stem cells (ADSC) to secrete various cytokines, which can be used for skin damage repair.

[0006] The patent CN110157666A of our team has confirmed that by adding the Chinese medicinal ingredients Chuanxiongqin hydrochloride and Shenmai injection and adopting the method of hypoxia culture during the culture process, the cell activity of mesenchymal stem cells (MSC) can be effectively improved and cell aggregation can be reduced, and the rouleaux-like aggregation of human red blood cells after reinfusion can be reduced. In subsequent studies, we found that Chuanxiongqin and Shenmai injection can also promote the synthesis and secretion of Wnt family members Wnt-1a by mesenchymal stem cells (MSC), and the addition of small molecule compounds all-trans retinoic acid (ATRA) and vitamin B2 in the culture can greatly promote the synthesis and secretion of proteins such as Wnt-1a, vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang-1), which can effectively promote the accumulation of β-ctenin protein in human hair follicle stem cells and promote the proliferation of hair follicle stem cells. It can effectively promote hair regeneration in human experiments.

[0007] Based on this, the present invention provides a mesenchymal stem cell hair growth liquid for hair loss caused by excessive secretion of male hormones. Summary of the invention

[0008] In order to solve at least one problem existing in the prior art, the present invention provides a mesenchymal stem cell hair growth liquid, which can effectively prevent and treat hair loss caused by excessive secretion of male hormones and promote human hair regeneration.

[0009] The present invention provides a mesenchymal stem cell hair growth fluid, comprising mesenchymal stem cells (MSCs), wherein the mesenchymal stem cells carry Wnt-1a protein and vasoactive factors, wherein the vasoactive factors include endothelial cell growth factor VEGF and angiopoietin Ang-1.

[0010] Among the more than 200 cytokines that mesenchymal stem cells can secrete, there is a large category that has a significant ability to promote angiogenesis, known as vasoactive factors, including vascular endothelial growth factor (VEGF), angiopoietin (Ang), fibroblast growth factor (bFGF), etc. Among them, vascular endothelial growth factor (VEGF) is the most important cell growth factor in the angiogenesis process. VEGF can directly stimulate the proliferation and migration of vascular endothelial cells in vitro. In the in vivo angiogenesis model, VEGF can be seen as a powerful angiogenesis inducing factor. In addition, VEGF also has the effect of inhibiting the apoptosis of vascular endothelial cells. It is the most important stimulating factor in the entire angiogenesis process. Angiopoietin (Ang) is a class of secretory cytokines that play an important role in vascular remodeling and embryonic vascular development. There are four members of the angiopoietin family, including Ang-1, Ang-2, Ang-3, and Ang-4. Among them, Ang-1 plays a major physiological role. Ang-1 inhibits endothelial cell apoptosis and promotes endothelial cell survival by activating its receptor tyrosine kinase tie-2, and further promotes endothelial cell sprouting, migration and chemotaxis to form new blood vessels. At the same time, it stabilizes the structure of new blood vessels and prevents wall leakage and other important physiological functions.

[0011] The Wnt signaling pathway plays an important role in maintaining the activity of DP cells. Wnt protein is a secretory protein of stem cells. It can increase the accumulation of β-catenin, an important protein that regulates gene expression, through cell-to-cell interactions and Wnt signaling pathways in target cells, thereby controlling the proliferation and differentiation of target cells. Studies have shown that during the formation and regeneration of hair follicles, β-catenin mRNA is upregulated and the Wnt signaling pathway is active in the dermis; and Wnt is only expressed in large quantities in hair follicles during the growth phase, indicating that the expression of Wnt and β-catenin is closely related to the formation and regeneration of hair follicles. The Wnt signal secreted by stem cells in the bulge of the hair follicle can maintain the activity of stem cells throughout the hair cycle, maintain the gene expression in the growth phase and the hair induction activity; and β-catenin activation can induce the proliferation of dermal fibroblasts and increase the thickness / density of the dermis; the proliferation of hair follicle cells is accelerated and the differentiation is accelerated. If the Wnt pathway is interrupted, for example, the gene expression of Wnt or β-catenin is inhibited, the morphogenesis and regeneration of hair follicles will be seriously affected: without Wnt, hair regeneration will be delayed, and hair follicles will be difficult to form during wound recovery; without β-catenin, the hair follicle inducing effect of Wnt disappears, resulting in hair loss during regression, hair follicle atrophy and inability to regenerate, and at the same time, the stem cells in the bulge of the hair follicle cannot differentiate into hair follicle stem cells, but can only differentiate into epidermal keratinocytes.

[0012] The shampoo of the present invention introduces mesenchymal stem cells carrying Wnt-1a protein and vasoactive factors through directed cultivation, and the stem cells can be used to effectively promote the proliferation and migration of vascular endothelial cells in vitro, and can effectively promote human hair regeneration through intradermal introduction, which can effectively prevent and treat hair loss and promote human hair regeneration.

[0013] Preferably, the mesenchymal stem cells are umbilical cord blood mesenchymal stem cells.

[0014] Preferably, the mesenchymal stem cells are obtained by culturing in a serum-free culture medium containing additives, and the effective ingredients contained in the additives are ligustrazine hydrochloride, shengmai injection, all-trans retinoic acid and riboflavin.

[0015] The above-mentioned serum-free culture medium additive, by compounding traditional Chinese medicine monomers and small molecule compounds, can effectively promote umbilical cord mesenchymal stem cells (UC-MSC) to express and secrete Wnt-1a molecules, as well as angiogenesis-related cell growth factors endothelial cell growth factor (VEGF), angiopoietin 1 (Ang-1), etc., and can effectively promote the accumulation of β-ctenin protein in human hair follicle stem cells and promote the proliferation of hair follicle stem cells. It can effectively promote hair regeneration when used in human experiments.

[0016] Preferably, in the serum-free culture medium containing additives, the concentration of ligustrazine hydrochloride is 40-80 mg / L; the volume percentage of shengmai injection is 0.5-1.0%, the concentration of all-trans retinoic acid is 0.1-50 mM, and the concentration of riboflavin is 0.05-50 mM.

[0017] Preferably, the serum-free medium containing additives is prepared by the following method, comprising the steps of:

[0018] (1) preparing a mother solution, wherein the mother solution comprises an all-trans retinoic acid solution, a riboflavin solution and a ligustrazine hydrochloride solution, wherein the solvent in the all-trans retinoic acid solution is DMSO, the solvent in the riboflavin solution is anhydrous ethanol, and the solvent in the ligustrazine hydrochloride solution is a phosphate buffer solution free of calcium / magnesium ions;

[0019] (2) Add the mother solution and Shenmai injection into common serum-free culture medium and mix well.

[0020] The present invention also provides a method for preparing the above-mentioned mesenchymal stem cell hair growth fluid, comprising the following steps:

[0021] S1 mesenchymal stem cell subculture;

[0022] S2 uses the serum-free medium containing additives to culture the mesenchymal stem cells after passage under hypoxia;

[0023] S3 cytokine supernatant collection.

[0024] Preferably, the step S1 is: culturing the P1 generation mesenchymal stem cells to the P4 generation using a common serum-free culture medium.

[0025] Preferably, the step S2 comprises: S21 taking P4 mesenchymal stem cells after subculture, inoculating, and then using the serum-free basal medium containing additives for low-oxygen culture;

[0026] S22: replacing the serum-free culture medium containing additives and performing hypoxic culture.

[0027] Preferably, in steps S21 and S22, the oxygen concentration of the hypoxic culture is 0.5% to 10%.

[0028] Preferably, the step S21 is: taking the P4 generation mesenchymal stem cells after the subculture, and calculating the number of living cells at 1-10*10 5 / cm 2 After inoculation, use ordinary serum-free basal culture medium and place in a low oxygen environment with an oxygen concentration of 0.5% to 10% for 24±1h; when the cells grow to 70% to 90% confluence, replace the serum-free culture medium containing additives at 0.1-0.5ml / cm 2 , hypoxic culture for 48±2h;

[0029] In step S22, the hypoxic culture time is 48±2h.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention provides a mesenchymal stem cell hair growth liquid and a preparation method thereof. By directional cultivation of mesenchymal stem cells, a method of combining traditional Chinese medicine monomers with hypoxia pretreatment is used to stimulate mesenchymal stem cells, thereby greatly improving the expression and secretion of Wnt-1a protein, vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang-1) in the product. Among them, Wnt-1a protein can activate dermal papilla (DP) cells through the Wnt / β-catenin signaling pathway, activate dormant hair follicle stem cells, and make dormant hair follicles active again, thereby promoting hair regeneration, while WEGF and Ang-1 can effectively promote subcutaneous angiogenesis, promote blood supply to hair follicles, and further promote hair regeneration. Therefore, the hair growth liquid product of the present invention has a significant effect in promoting hair regeneration, and can be used to prevent and treat hair loss caused by vigorous secretion of androgen. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The β-catenin protein content test results in Experimental Example 1;

[0033] Figure 2 This is the EDU detection test result in Experimental Example 1. DETAILED DESCRIPTION

[0034] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0036] The reagents used in the following examples, unless otherwise specified, are all commercially available; the methods used in the following examples, unless otherwise specified, are all achievable by conventional methods.

[0037] Example 1

[0038] Preparation of serum-free medium with supplements:

[0039] ⑴ Prepare mother liquor,

[0040] Specifically, the solvent dimethyl sulfoxide (DMSO) is used to prepare the all-trans retinoic acid solution, anhydrous ethanol is used to dissolve riboflavin to obtain a riboflavin solution, and a phosphate buffer solution without calcium / magnesium ions is used to prepare the ligustrazine hydrochloride solution.

[0041] ⑵ The mother solution prepared in step (1) and Shenmai injection are added to a common serum-free medium (purchased from Beijing Youkang Hengye Biotechnology Co., Ltd. NC0103 / NC0105.S) and mixed to obtain a serum-free medium containing additives, wherein the concentration of ligustrazine hydrochloride is 60 mg / L, the concentration of all-trans retinoic acid is 10 mM, the concentration of riboflavin (VB2) solution is 5 mM, and the volume percentage of Shenmai injection is 0.5%.

[0042] Example 2

[0043] A mesenchymal stem cell hair growth liquid comprises mesenchymal stem cells (MSCs), wherein the mesenchymal stem cells carry Wnt-1a protein and vasoactive factors, wherein the vasoactive factors include endothelial cell growth factor VEGF and angiopoietin Ang-1. The preparation method of the hair growth liquid comprises the following steps:

[0044] S1 mesenchymal stem cell subculture;

[0045] Umbilical cord mesenchymal stem cells (UC-MSC) of the P1 generation were revived according to conventional methods and cultured to the P4 generation using serum-free basal medium (NC0103 / NC0105.S, Beijing Youkang Hengye Biotechnology Co., Ltd.);

[0046] S2 uses the serum-free medium containing additives to culture the mesenchymal stem cells after passage under hypoxia;

[0047] The umbilical cord mesenchymal stem cells after the above subculture were taken and the cells were divided into 10×10 5 / cm 2 The cells were inoculated at a density of 100 μg / mL and cultured for 24 ± 1 h in a hypoxic environment with an oxygen concentration of 0.5% to 10% using serum-free basal medium (NC0103 / NC0105.S from Beijing Youkang Hengye Biotechnology Co., Ltd.).

[0048] When the cells grow to 70% to 90% confluence, replace the medium with serum-free medium containing additives, place the cells in a low oxygen environment with an oxygen concentration of 0.5% to 10%, and culture for 48±2h.

[0049] S3 cytokine supernatant collection.

[0050] Experimental Example 1

[0051] A mesenchymal stem cell hair growth liquid, the preparation method of the hair growth liquid comprises the steps of:

[0052] 1. Subculture: Resuscitate P1 umbilical cord mesenchymal stem cells (UC-MSC) according to conventional methods, and culture them normally to P4 using serum-free basal medium (Beijing Youkang Hengye Biotechnology Co., Ltd. NC0103 / NC0105.S);

[0053] 2. Hypoxia culture:

[0054] When the P4 generation UC-MSCs grew to 90% confluence, they were digested into single cells using mild digestive enzymes (NC1004.1 from Beijing Youkang Hengye Biotechnology Co., Ltd.), counted, and centrifuged (1500 rpm × 5 min), and the supernatant was removed;

[0055] According to the counting results, the number of living cells was calculated as 2×10 5 / cm 2 The cells were inoculated into 6-well plates at a density of 1.50 μg / mL and cultured in a low oxygen (oxygen content 5%, carbon dioxide content 5%) incubator for 24 hours using serum-free basal medium (Beijing Youkang Hengye Biotechnology Co., Ltd. NC0103 / NC0105.S).

[0056] When the cells grew to 90% confluence, the cells were randomly divided into 2 groups and the culture medium was changed: the normal group was cultured with serum-free basal medium, and the stimulation group was cultured with serum-free medium prepared in Example 1, 2 mL was added to each well, and each group was cultured for 48 hours in a hypoxic environment (oxygen content of 5%, carbon dioxide content of 5%);

[0057] 3. Collection of cytokine supernatant

[0058] After 48 hours, the cytokines of each group were collected and frozen at -20°C. The cells were washed with PBS and resuspended in Trizol (Thermo 15596026). Total RNA was extracted from the cells and reversely transcribed into cDNA and stored at -20°C (Tiangen KR106).

[0059] Test: The expression level of the active ingredient wnt-1a in each group of cells was detected by QPCR (Promega A6002); the primer sequences used for QPCR are shown in Table 1.

[0060] Table 1 Primer sequences used for QPCR

[0061] Serial number name Primer sequences SEQ ID NO:1 Wnt-1a 5'-GGTTTCTACTACGTTGCTACTGG-3' SEQ ID NO:2 Wnt-1a 5'-GGAATCCGTCAACAGGTTCGT-3' SEQ ID NO:3 VEGF 5'-GACCCTGGTGGACATCTTCC-3' SEQ ID NO:4 VEGF 5'-CACAGGGATTTTCTTGTCTTGC-3' SEQ ID NO:5 Ang-1 5'-CGGTGAATATTGGCTGGGGAATGAG-3' SEQ ID NO:6 Ang-1 5'-GTAGTGCCACTTTATCCCATTCAG-3'

[0062] The collected cytokine supernatants were used to detect the active components secreted by cells in each group using the Ellisa kit, including Wmt-1a protein (ENZO-1ASB-OKEH04852), vascular endothelial cell growth factor (VEGF) (Xinbosheng EHC108), and angiopoietin 1 (Ang-1) (ELISA MM-0001H2). The test results are shown in Table 2: The expression level of Wnt-1a gene in the stimulation group was 1 million times that of the normal group (1×10 6 ), the secretion of Wnt-1a protein in the supernatant was 321.25 times that of the normal group (21.14 vs 6791.23); the expression of VEGF gene in the stimulation group was 10000 times that of the normal group, and the VEGF gene in the supernatant was about 176 times that of the normal group (18.62 vs 3276.48); the expression of Ang-1 gene in the stimulation group was 100 times that of the normal group, and the Ang-1 gene in the supernatant was about 45 times that of the normal group (19.21 vs 867.55); It can be seen that under the stimulation pretreatment of traditional Chinese medicine monomers, MSC can greatly increase the expression and secretion of Wnt-1a, VEGF, and Ang-1 proteins.

[0063] Table 2 Wnt-1a expression test results

[0064]

[0065]

[0066] Experimental Example 2

[0067] A mesenchymal stem cell hair growth liquid, the preparation method of the hair growth liquid comprises the steps of:

[0068] 1. Subculture: Resuscitate P1 umbilical cord mesenchymal stem cells (UC-MSC) according to conventional methods, and culture them normally to P4 using serum-free basal medium (Beijing Youkang Hengye Biotechnology Co., Ltd. NC0103 / NC0105.S);

[0069] 2. Hypoxia culture:

[0070] When the P4 generation UC-MSCs grew to 90% confluence, they were digested into single cells using mild digestive enzymes (NC1004.1 from Beijing Youkang Hengye Biotechnology Co., Ltd.), counted, and centrifuged (1500 rpm × 5 min), and the supernatant was removed;

[0071] According to the counting results, the number of live cells was inoculated into a 6-well plate at a density of 2 × 105 / cm2 and cultured in a hypoxic (oxygen content 5%, carbon dioxide content 5%) incubator for 24 h using serum-free basal medium (NC0103 / NC0105.S, Beijing Youkang Hengye Biotechnology Co., Ltd.);

[0072] When the cells grew to 90% confluence, they were randomly divided into 3 groups and the culture medium was changed: the normal group was cultured with serum-free basal medium, and the stimulation group was cultured with serum-free basal medium + 60 mg / L ligustrazine hydrochloride + 0.5% Shenmai injection + 50 μM vitamin C, 2 mL was added to each well, and each group was cultured in a hypoxic environment (oxygen content of 5%, carbon dioxide content of 5%) for 48 hours;

[0073] 3. Collection of cytokine supernatant

[0074] The cytokine supernatant was collected, concentrated 10 times by centrifugation in an ultrafiltration tube (Millipore UFC900396), and refrigerated for later use.

[0075] 4. Human hair follicle stem cells were cultured to P2 generation using a special culture medium at 20,000 / cm 2 The density of was inoculated in a 6-well plate and cultured overnight;

[0076] 5. Hair follicle stem cells were divided into two groups. The first group was a normal group (special culture medium for hair follicle stem cells: PBS = 1:1), and the second group was an experimental group (special culture medium for hair follicle stem cells: concentrated supernatant = 1:1). After normal culture for 48 hours, cells from each group were taken for Wersten Blot detection of β-catenin protein content and EDU detection of cell proliferation activity. The results are as follows: Figure 1 and Figure 2As shown, the supernatant of the experimental group can effectively promote the accumulation of β-catenin protein in human hair follicle stem cells and activate downstream genes, thereby promoting the proliferation of hair follicle stem cells.

[0077] 6. Nine patients with hair loss caused by excessive androgen secretion were screened. The cell supernatant concentrate was sterilized by a 0.22μm filter membrane and then introduced into the sparse hair area of ​​the scalp by a water light needle method. The needle insertion depth was 0.8mm, and the scalp was red but not bleeding. The treatment was once a week for 4 consecutive weeks as a course of treatment. In the 4th and 12th weeks, a hair mirror was used to observe the hair regeneration before and after administration. The pre-treated MSC supernatant can effectively promote the hair regeneration of patients. The hair density before treatment was 44.33 hairs / cm 2 After 12 weeks of use, the hair density was 73.67 cm 2 , as shown in Table 3 below.

[0078] Table 3 Hair growth effect

[0079]

[0080]

[0081] This example also verifies through in vivo use that the hair growth liquid of the present invention can effectively promote the accumulation of β-ctenin protein in human hair follicle stem cells and activate downstream genes, thereby promoting the proliferation of hair follicle stem cells and effectively promoting hair regeneration.

[0082] In summary, it can be seen that the present invention stimulates mesenchymal stem cells by directed cultivation of mesenchymal stem cells using traditional Chinese medicine monomers combined with hypoxic pretreatment, thereby greatly improving the expression and secretion of Wnt-1a protein, vascular endothelial growth factor (VEGF), and angiopoietin 1 (Ang-1) in exosomes. The obtained hair growth liquid product can effectively promote human hair regeneration through intradermal introduction, and can be used to prevent and treat hair loss caused by excessive secretion of androgen.

[0083] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0084] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A mesenchymal stem cell hair growth fluid, characterized in that: The invention comprises mesenchymal stem cells, wherein the mesenchymal stem cells carry Wnt-1a protein and vasoactive factors, and the vasoactive factors include endothelial cell growth factor VEGF and angiopoietin Ang-1.

2. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: The mesenchymal stem cells are umbilical cord blood mesenchymal stem cells.

3. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: The mesenchymal stem cells are obtained by culturing a serum-free culture medium containing additives, wherein the effective ingredients contained in the additives are ligustrazine hydrochloride, shengmai injection, all-trans retinoic acid and riboflavin.

4. The mesenchymal stem cell hair growth fluid according to claim 3, characterized in that: In the serum-free culture medium containing additives, the concentration of ligustrazine hydrochloride is 40-80 mg / L, the volume percentage of shengmai injection is 0.5-1.0%, the concentration of all-trans retinoic acid is 0.1-50 mM, and the concentration of riboflavin is 0.05-50 mM.

5. The mesenchymal stem cell hair growth fluid according to claim 4, characterized in that: The serum-free medium containing additives is prepared by the following method, comprising the steps of: (1) preparing a mother solution, wherein the mother solution comprises an all-trans retinoic acid solution, a riboflavin solution and a ligustrazine hydrochloride solution, wherein the solvent in the all-trans retinoic acid solution is DMSO, the solvent in the riboflavin solution is anhydrous ethanol, and the solvent in the ligustrazine hydrochloride solution is a phosphate buffer solution free of calcium / magnesium ions; (2) Add the mother solution and Shenmai injection into common serum-free culture medium and mix well.

6. The method for preparing the mesenchymal stem cell hair growth liquid according to any one of claims 1 to 5, characterized in that: The following steps are involved: S1 mesenchymal stem cell subculture; S2 uses the serum-free medium containing additives to culture the mesenchymal stem cells after passage under hypoxia; S3 cytokine supernatant collection.

7. The method for preparing the mesenchymal stem cell hair growth liquid according to claim 6, characterized in that: The step S1 is: culturing the P1 generation mesenchymal stem cells to the P4 generation using a common serum-free culture medium.

8. The method for preparing the mesenchymal stem cell hair growth liquid according to claim 7, characterized in that: The step S2 comprises: S21 taking the P4 generation mesenchymal stem cells after subculture, inoculating them, and then using the serum-free basal medium containing additives to perform hypoxia culture; S22: replacing the serum-free culture medium containing additives and performing hypoxic culture.

9. The method for preparing the mesenchymal stem cell hair growth liquid according to claim 8, characterized in that: In the steps S21 and S22, the oxygen concentration of the hypoxic culture is 0.5% to 10%.

10. The method for preparing the mesenchymal stem cell hair growth liquid according to claim 9, characterized in that: The step S21 is: taking the P4 generation mesenchymal stem cells after the above subculture, and calculating the number of living cells at 1-10*10 5 / cm 2 After inoculation, use ordinary serum-free basal culture medium and place in a low oxygen environment with an oxygen concentration of 0.5% to 10% for 24±1h; when the cells grow to 70% to 90% confluence, replace the serum-free culture medium containing additives at 0.1-0.5ml / cm 2 , hypoxic culture for 48±2h; In step S22, the hypoxic culture time is 48±2h.

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