A pharmaceutical composition for improving cell therapy regeneration and its application

Abstract

The embodiment of the present invention discloses a pharmaceutical composition for improving the regeneration of cell therapy and its application. The pharmaceutical composition includes: 5-15ng / mL transforming growth factor β, 0.02-0.06μM triptolide, 8-12ng / mL vascular endothelial growth factor, 0.1-1μg / mL grape seed extract, 0.8-1.6mg / mL trace element solution, 0.5-2% hyaluronic acid, 0.05-0.15μM cyclosporine, 15-25mM HEPES buffer. The pharmaceutical composition provided by the present invention can effectively promote the proliferation and differentiation of hair follicle-derived mesenchymal stem cells, play a good role in treating hair loss, and has small side effects, high safety, low cost, wide application population, and good application prospects in clinical practice.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and more particularly to a pharmaceutical composition for improving cell therapy regeneration and its application. Background Art

[0002] In the field of hair loss treatment, existing methods mainly include drug therapy, hair transplant surgery, and other auxiliary treatments. Drug treatments such as minoxidil and finasteride are commonly used to treat male pattern baldness and can promote hair growth or slow hair loss, but minoxidil requires long-term use and its effects may disappear after discontinuation; while finasteride is effective, it may cause side effects. The combined use of these two drugs may enhance the treatment effect, but the applicable population is usually limited to patients with early-stage hair loss. Hair transplant surgery, such as hair follicle transplantation, can restore hair growth with obvious and long-lasting effects, but it is costly and the surgery is complicated. It is suitable for patients with large areas of hair loss or who have not responded well to drug treatment. Other auxiliary treatment methods such as laser treatment, scalp massage and herbal treatment each have their own advantages. For example, laser treatment is non-invasive but the effect varies from person to person, scalp massage is simple and easy but the effect is limited, and herbal treatment is natural and has no side effects, but the effect requires long-term observation.

[0003] Cell therapy utilizes the cells' inherent regenerative capacity to promote hair growth rather than simply inhibiting hair loss. Cell therapy mimics the natural tissue repair process by introducing healthy cells to repair damaged hair follicles, rather than relying on external drugs or chemicals. Unlike some traditional therapies that can create dependency, cell therapy has the potential to provide a long-term solution because it relies on the cells' inherent function and regenerative capacity. Cell therapy can be personalized to each patient's specific situation to enhance therapeutic effectiveness. Because cell therapy uses the patient's own or allogeneic cells, it has fewer side effects than some traditional therapies. However, the success of cell therapy ultimately depends on the number of cells that reach the target tissue, their viability, and their promotion of tissue regeneration. This key factor determines the effectiveness and long-term effectiveness of cell therapy in regenerative medicine.

[0004] A sufficient cell count ensures that enough cells will colonize the hair follicles and initiate new hair growth. Insufficient cell counts may not cover all damaged follicle areas, compromising the overall treatment effect. Highly active cells are better able to adapt to the hair follicle environment, carry out necessary metabolic activities, secrete growth factors, and promote hair follicle recovery and hair growth. By secreting growth factors and extracellular matrix components, cells activate and promote the regeneration process of hair follicles. These factors stimulate the hair follicles to transition from the resting phase to the growing phase, thereby promoting hair growth.

[0005] Therefore, when cell therapy is used to treat hair loss, cell quantity, vitality, and the promotion of tissue regeneration are key factors to ensure the effectiveness of treatment. Only when these conditions are met can cell therapy effectively promote hair growth and achieve the goal of treating hair loss. Summary of the Invention

[0006] To this end, embodiments of the present invention provide a pharmaceutical composition for improving regeneration in cell therapy and its application.

[0007] In order to achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of an embodiment of the present invention, the present invention provides a pharmaceutical composition for improving regeneration in cell therapy, the pharmaceutical composition comprising: 5-15 ng / mL transforming growth factor β, 0.02-0.06 μM triptolide, 8-12 ng / mL vascular endothelial growth factor, 0.1-1 μg / mL grape seed extract, 0.8-1.6 mg / mL trace element solution, 0.5-2% hyaluronic acid, 0.05-0.15 μM cyclosporine, and 15-25 mM HEPES buffer.

[0009] Transforming Growth Factor-β: TGF-β is an important cytokine that plays a crucial role in biological processes such as cell growth, differentiation, apoptosis, and extracellular matrix synthesis. The combined use of TGF-β and hair follicle-derived mesenchymal stem cells can enhance the growth and differentiation of hair follicle-derived mesenchymal stem cells through the stimulating effect of TGF-β, thereby accelerating new hair growth and improving therapeutic efficacy. The transforming growth factor-β used in this embodiment is from Okey Biotech (Cat. No. P1230).

[0010] Triptolide (CAS No. 38748-32-2) is a diterpene lactone compound extracted from the root of the traditional Chinese medicine Tripterygium wilfordii. It exhibits significant immunosuppressive, anti-inflammatory, and anti-tumor activities. A growing number of studies have demonstrated its significant efficacy in the treatment of inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, and nephritis. Triptolide's anti-inflammatory mechanism primarily regulates the activity of key proteins in the inflammatory signaling pathway, including MAPK, PI3K, Jak2, Bc1-2 / Bax8, JNK-1 / 2, MPK1, ERK-1 / 2, NF-KB, activated ROS, and caspase 8, thereby inhibiting nitric oxide production and COX-2 expression, and downregulating the expression of inflammatory cytokines such as TNF-α, IL-1β, and IL-6.

[0011] Vascular endothelial growth factor (VEGF) (CAS No. 127464-60-2): also known as vascular permeability factor, angiostatic factor (VAS), or vasomodulin. It is purified from the culture medium of pituitary stellate follicle cells and ovarian cells of various rodents. It is a specific growth factor associated with angiogenesis and promotes vascular permeability. VEGF binds to specific receptors (Flt-1, Flk-1 / KDR) on the surface of endothelial cells, promoting endothelial cell proliferation and stimulating angiogenesis in the body. Furthermore, as a local endogenous regulator, VEGF plays a role in maintaining the normal state and integrity of blood vessels.

[0012] Grape seed extract: Rich in polyphenolic compounds, such as proanthocyanidins and resveratrol, these ingredients possess potent antioxidant properties. They can scavenge free radicals in the body, reduce oxidative damage to cells, and thus protect cardiovascular and cerebrovascular systems. The grape seed extract used in this example was obtained from Shaanxi Panier Biotechnology Co., Ltd. (Cat. No. PB20211217).

[0013] Trace elements play a crucial role in hair loss, playing a vital role in hair growth and health. Their primary role in hair loss is to maintain the hair's growth environment, promote healthy hair growth, and prevent hair loss. Maintaining appropriate trace element levels is crucial for preventing and treating hair loss. The trace element solution used in this embodiment of the present invention is from Shanghai Huzhen Industrial Co., Ltd. (Article No. HZ1941-50L) and consists of: 50 μg CuSO₄, 16 μg MnSO₄, 30 μg ZnSO₄, 220 μg CoCl₂ 6H₂O, and 45 μg FeSO₄, diluted to 100 ml with water.

[0014] Hyaluronic acid (CAS No. 9004-61-9): A major component of connective tissues such as the human intercellular matrix, vitreous humor, and synovial fluid, it plays an important role in the body in retaining water, maintaining the extracellular space, regulating osmotic pressure, providing lubrication, and promoting cell repair. As a major component of the intercellular matrix, hyaluronic acid directly regulates the flow of electrolytes inside and outside cells, acting as a filter for physical and molecular information. The hyaluronic acid used in the examples of this invention is sourced from Tonghui Pharmaceutical.

[0015] Cyclosporine (CAS No. 59865-13-3): An immunosuppressant commonly used to prevent organ rejection after transplantation and to treat some autoimmune diseases. Its purpose is to reduce rejection and help newly implanted hair follicles grow steadily.

[0016] HEPES buffer (CAS No. 7365-45-9): The optimal pH for the scalp is typically between 4.5 and 5.5. This pH range helps maintain the scalp's natural protective barrier and keeps it healthy. The scalp's acidic environment helps prevent the growth of bacteria and fungi, reducing dandruff and hair problems.

[0017] Furthermore, the pharmaceutical composition includes: 10 ng / mL transforming growth factor β, 0.04 μM triptolide, 10 ng / mL vascular endothelial growth factor, 0.5 μg / mL grape seed extract, 1 mg / mL trace element solution, 1% hyaluronic acid, 0.1 μM cyclosporine, and 20 mM HEPES buffer.

[0018] According to a second aspect of an embodiment of the present invention, the present invention provides a method for preparing a pharmaceutical composition for improving cell therapy regeneration as described in any of the above items, wherein transforming growth factor β, triptolide, vascular endothelial growth factor, grape seed extract, trace element solution, hyaluronic acid, cyclosporine and HEPES buffer are dissolved in DMEM / F12 and mixed to obtain the pharmaceutical composition for improving cell therapy regeneration.

[0019] According to a third aspect of the embodiments of the present invention, the present invention provides a method for improving regeneration in cell therapy, the method comprising: culturing hair follicle-derived mesenchymal stem cells using the pharmaceutical composition as described in any one of the above items.

[0020] Furthermore, the method comprises:

[0021] (1) Cultivate hair follicle tissue, subculture cells when they reach 80%-90% confluence, and then obtain hair follicle-derived mesenchymal stem cells through digestion, pipetting, inoculation, culture observation, and identification;

[0022] (2) When the hair follicle-derived mesenchymal stem cells reach the 3rd to 5th passages and the cells reach a confluence of 80% to 90%, the drug composition is added and cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0023] Furthermore, in step (1), the culture and subculture conditions are: 37°C, 5% CO2 and 95% air, and the culture medium is DMEM / F12 containing 10v / v% fetal bovine serum.

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

[0025] According to a fifth 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.

[0026] The embodiments of the present invention have the following advantages:

[0027] Solve the problems of long medication time and poor effect of existing drug treatments;

[0028] Solve the problems of many side effects of existing drug treatments;

[0029] Solve the problems of existing surgical treatment methods such as high cost, only autologous transplantation, slow postoperative recovery, and many complications;

[0030] Solve the problem that existing treatment methods are only applicable to a single group of people and cannot effectively treat various hair loss patients;

[0031] Solve the problem of massive cell death in a short period of time after cell transplantation due to lack of nutrients and oxygen in the recipient site and host inflammatory response. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the embodiments or the description of the prior art. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other implementation drawings based on the provided drawings without inventive effort.

[0033] Figure 1 The cell proliferation results of the hair follicle-derived mesenchymal stem cells provided by the present invention at the 7th and 11th days of culture;

[0034] Figure 2 The survival test results of each group of cells provided by the present invention under different induction environments;

[0035] Figure 3 The number detection results of each group of cells under different induction environments provided by the present invention;

[0036] Figure 4 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0037] Figure 5 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0038] Figure 6 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0039] Figure 7 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0040] Figure 8 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0041] Figure 9 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0042] Figure 10 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0043] Figure 11 The comparison results of hair growth of a volunteer before and after treatment provided by the present invention;

[0044] Figure 12 The present invention provides a comparison of the hair growth results of a volunteer before and after treatment. DETAILED DESCRIPTION

[0045] The following describes the implementation of the present invention using specific embodiments. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. Obviously, the embodiments described are only a portion of the present invention, not all of it. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0046] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] Example 1

[0048] This example provides a pharmaceutical composition for enhancing regeneration in cell therapy, comprising: 10 ng / mL transforming growth factor β, 0.04 μM triptolide, 10 ng / mL vascular endothelial growth factor, 0.5 μg / mL grape seed extract, 1 mg / mL trace element solution, 1% hyaluronic acid, 0.1 μM cyclosporine, and 20 mM HEPES buffer, using DMEM / F12 as the solvent.

[0049] Example 2

[0050] This example provides a pharmaceutical composition for enhancing regeneration in cell therapy, comprising: 5 ng / mL transforming growth factor β, 0.02 μM triptolide, 12 ng / mL vascular endothelial growth factor, 0.1 μg / mL grape seed extract, 1.4 mg / mL trace element solution, 0.5% hyaluronic acid, 0.12 μM cyclosporine, and 16 mM HEPES buffer, using DMEM / F12 as the solvent.

[0051] Example 3

[0052] This example provides a pharmaceutical composition for enhancing regeneration in cell therapy, comprising: 14 ng / mL transforming growth factor β, 0.06 μM triptolide, 8 ng / mL vascular endothelial growth factor, 0.8 μg / mL grape seed extract, 0.9 mg / mL trace element solution, 1.5% hyaluronic acid, 0.06 μM cyclosporine, and 22 mM HEPES buffer, using DMEM / F12 as the solvent.

[0053] Comparative Example 1

[0054] This comparative example provides a pharmaceutical composition for improving regeneration in cell therapy, which comprises: 10 ng / mL fibroblast growth factor, 0.05 μM triptolide, 10 ng / mL vascular endothelial growth factor, 0.5 μg / mL grape seed extract, 1 mg / mL trace element solution, 2% hyaluronic acid, 0.1 μM cyclosporine, and 20 mM HEPES buffer, with DMEM / F12 as the solvent.

[0055] Comparative Example 2

[0056] This comparative example provides a pharmaceutical composition for improving regeneration in cell therapy, which is composed of: 25 ng / mL transforming growth factor β, 10 ng / mL vascular endothelial growth factor, 0.5 μg / mL grape seed extract, 1 mg / mL trace element solution, 2% hyaluronic acid, 0.1 μM cyclosporine, and 20 mM HEPES buffer, with DMEM / F12 as the solvent.

[0057] Comparative Example 3

[0058] This comparative example provides a pharmaceutical composition for improving regeneration in cell therapy, comprising: 5 ng / mL transforming growth factor β, 0.01 μM triptolide, 15 ng / mL vascular endothelial growth factor, 2 μg / mL grape seed extract, 1.2 mg / mL trace element solution, 1.5% hyaluronic acid, 0.2 μM cyclosporine, and 16 mM HEPES buffer, using DMEM / F12 as a solvent.

[0059] Example 4

[0060] This embodiment provides a method for improving regeneration in cell therapy, comprising:

[0061] (1) Acquisition of hair follicle-derived mesenchymal stem cells

[0062] 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, 1 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.

[0063] In vitro culture: Cut the extracted hair follicle tissue into small pieces and evenly add them to the wells of the plate using complete culture medium. Continue culturing in a cell culture incubator at 37°C and 5% CO2. After 48 hours, replace half of the medium. Continue observing for 7-10 days to see if stem cells migrate out of the tissue pieces.

[0064] Passaging: When cells reach approximately 80% confluence, they are ready for passaging.

[0065] Digest cells: Aspirate the culture medium from the flask and wash the cells once with PBS. Then, add an appropriate amount of trypsin digestion solution (such as 0.25% trypsin) and incubate at 37°C for approximately 3 minutes. Observe under an inverted microscope. Once the cells shrink and become rounded, aspirate the digestion solution and add complete culture medium to terminate the digestion.

[0066] Pipetting and inoculating: Gently pipette the cells to dislodge them from the flask walls and disperse them into a single-cell suspension. Count and subculture the cells. Add 15 ml of fresh complete medium and continue culturing in a cell culture incubator at 37°C and 5% CO2.

[0067] Culture observation: After the cells have fully adhered, culture and observe. Replace with fresh complete culture medium after 48 hours to maintain a good growth environment for the cells.

[0068] Identification: Morphological characteristics are used for identification. Under a light microscope, hair follicle-derived mesenchymal stem cells usually appear as adherent cells with a long spindle-shaped morphology.

[0069] In vitro culture: The third generation of hair follicle-derived mesenchymal stem cells were seeded on a 35 mm culture dish at a density of 2×10 5 / cm 2 , cultured in an incubator at 37°C, 5% CO2 and 95% air until the cells were 80%-90% full-length.

[0070] (2) Cell culture: The culture medium supernatant in the culture dish was aspirated, and the cells were washed three times with PBS buffer. The residual PBS buffer was aspirated, and the obtained hair follicle-derived mesenchymal stem cells were mixed with any of the pharmaceutical compositions in Examples 1-3 at a ratio of 1*10^7 / ml, and cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2.

[0071] Test Case

[0072] 1. Cell photos

[0073] Figure 1 This study presents microscopic observations (4X magnification) of cell proliferation of hair follicle-derived mesenchymal stem cells isolated from hair follicle tissue on days 7 and 11 of culture. Culture conditions included 37°C, 5% CO2, and 95% air, using DMEM / F12 medium supplemented with 10% v / v fetal bovine serum. The results showed that the cells were polygonal or star-shaped, relatively small, and densely packed.

[0074] 2. Cell survival under different induction environments

[0075] Figure 2 Figures AH show the survival of each group of cells under different induction environments.

[0076] The cells are grouped as follows:

[0077] A is the normal group, 1*10^7 / ml hair follicle-derived mesenchymal stem cells were cultured in 10% fetal bovine serum + DMEM / F12 medium in a cell culture incubator at 37°C, 5% CO2, and 95% air for 72 hours.

[0078] B is the control group, 1*10^7 / ml hair follicle-derived mesenchymal stem cells were cultured in DMEM / F12 medium in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0079] C is experimental group 1, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of Example 1 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0080] D is experimental group 2, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of Example 2 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0081] E is experimental group 3, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of Example 3 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0082] F is control group 1, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of comparative example 1 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0083] G is control group 2, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of comparative example 2 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0084] H is the control group 3, 1*10^7 / ml hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of comparative example 3 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours.

[0085] The survival status of cells in each group was observed by inverted microscope. The experimental results showed that the cells in the normal group cultured showed a fish-like or whirlpool-like growth morphology, showing good cell aggregation and interaction characteristics (see Figure 2 A). However, under serum-free culture conditions, cells showed significant apoptosis and necrosis, as evidenced by the formation of intracellular vesicles and a significant decrease in cell membrane integrity, suggesting that the cells suffered a severe stress response in an environment lacking growth factors (see Figure 2 B). Apoptosis and necrosis were also observed in experimental groups 1-3 and control groups 1-3. However, the proportion of necrosis and apoptosis in experimental group 1 was significantly lower than in the other groups, and the integrity of its cell membrane was significantly better than in the other groups. This result indicates that the culture conditions in experimental group 1 provided a more favorable survival environment, effectively slowing the process of apoptosis and necrosis, providing important clues for subsequent research on cell survival mechanisms.

[0086] 3. Cell proliferation experiment in hypoxic environment

[0087] The cells were divided into 35 subgroups (7 experimental groups × 5 time point groups), each subgroup contained the same number of cells, and each group was configured with three independent replicate wells. A six-well cell culture plate was used, and 1×10^6 hair follicle-derived mesenchymal stem cells were inoculated in each well, and static culture was performed at 37°C and 5% carbon dioxide concentration for 6 hours. After the cells completed adherent growth, the culture conditions of each group were changed, among which the experimental groups 1-3 used the drug compositions of Examples 1-3 respectively, and the control groups 1-3 used the drug compositions of Comparative Examples 1-3 respectively. The control group used DMEM / F12, and then all cell subgroups were placed in a hypoxic incubator to maintain appropriate hypoxic conditions (1% O2). The number of cells in each subgroup was measured using a cell counter at 12 hours, 1 day, 3 days, 5 days, and 7 days respectively. The results are shown in Tables 1 and Figure 3 .

[0088] Table 1

[0089]

[0090] The results showed that within 12 hours to 3 days after cell attachment, the cell number in experimental group 1 increased, reaching 1.86 times the pre-seeding number, but did not achieve exponential growth. Starting on day 3, cells began to undergo apoptosis and necrosis, and by day 7, the cell number had dropped to 1.1 times the pre-seeding number. This result suggests that the induction environment in experimental group 1 promoted good cell growth in the early stages. Although the number of surviving cells decreased over time, it remained within the pre-seeding cell number, indicating that this induction environment was able to maintain cell survival under hypoxic and low-nutrient conditions. In contrast, the cell number in the control group continued to decline from 12 hours to 7 days after cell attachment, reaching 1 / 15 of the pre-seeding number by day 7. The cell number in experimental groups 2 and 3 remained largely unchanged from 12 hours to 3 days after cell attachment, but from day 3, cells began to undergo continuous apoptosis and necrosis. By day 7, the cell number in experimental group 2 had dropped to 5 / 6 of the pre-seeding number, while the cell number in experimental group 3 had dropped to 1 / 3 of the pre-seeding number. The cell number of control group 1 remained basically unchanged from 12 hours to 1 day after cell attachment, but from the first day onwards, cells showed continuous apoptosis and necrosis. By the 7th day, the cell number of control group 1 dropped to 3 / 20 of that before inoculation. The cell number of control groups 2 and 3 continued to decrease from 12 hours to 7 days after cell attachment. By the 7th day, the cell number of control group 2 dropped to 1 / 10 of that before inoculation, and the cell number of control group 3 dropped to 1 / 20 of that before inoculation (e.g. Figure 3 ).

[0091] 4. Changes in cell survival

[0092] The cells were divided into 7 groups, namely control group, experimental group 1, experimental group 2, experimental group 3, control group 1, control group 2 and control group 3. At least three wells were assigned to each group in a six-well plate, and 1*10^6 cells were plated in each well. The corresponding culture medium was added, and serum was added to the culture medium. The six-well plate was placed in a culture flask, and an appropriate amount of liquid was added to the culture flask to ensure that the six-well plate adhered to the wall. Incubate in the culture box for 2-4 hours to allow the cells to adhere. The six-well plate was removed from the culture box and transferred to a hypoxic culture environment with an oxygen concentration of 1%. Cultured in a hypoxic environment for three days. At the end of the experiment, the number of cells, the number of viable cells, and the agglomeration rate in each well were counted and analyzed. The results are shown in Table 2 below.

[0093] Table 2

[0094]

[0095] Analysis of experimental data showed that under different induction drug culture conditions, only the concentration of live cells in experimental group 1 showed a significant increase, the cell survival rate exceeded 50%, and its clumping rate was maintained at the lowest level. In contrast, the number of live cells in other groups decreased significantly, while the cell clumping rate increased significantly. Specifically, the cell clumping rates of the control group, experimental group 3, and control group 1-2 all increased to more than 50%, among which the cell clumping rate of control group 1 was as high as 70.33%. This shows that the induction scheme adopted by experimental group 1 can not only effectively promote cell proliferation in a hypoxic environment, but also significantly reduce the cell clumping rate, thereby reducing cell death.

[0096] 5. In order to further explore the therapeutic potential of the method of the present invention and verify its safety and effectiveness in improving hair loss, we recruited 9 volunteers with hair loss. The induced hair follicle-derived mesenchymal stem cells (the third-generation hair follicle-derived mesenchymal stem cells and the pharmaceutical composition of Example 1 were cultured in a cell culture incubator at 37°C, 1% O2, 5% CO2, and 94% N2 for 72 hours) were injected into the scalp. The treatment frequency was once every 4 weeks, for a total of 3 treatments. The efficacy evaluation was analyzed using head hair images. The experimental results showed that the number of hair growth in the treatment area of ​​the 8 patients increased significantly, and the proportion of hair in the growth phase increased (such as Figure 4-11 As shown, the left picture is before treatment, and the right picture is after treatment). In addition, after three treatments, the amount of hair growth of one hair loss patient increased, but the effect was not very obvious (as shown in the figure). Figure 12 , the left picture is before treatment, and the right picture is after treatment). During the study, no adverse reactions were observed.

[0097] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made thereto. Therefore, such modifications and improvements, without departing from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A pharmaceutical composition for promoting the proliferation of hair follicle-derived mesenchymal stem cells, characterized in that: The pharmaceutical composition consists of the following components: 5-15 ng / mL transforming growth factor β, 0.02-0.06 μM triptolide, 8-12 ng / mL vascular endothelial growth factor, 0.1-1 μg / mL grape seed extract, 0.8-1.6 mg / mL trace element solution, 0.5-2% hyaluronic acid, 0.05-0.15 μM cyclosporine, and 15-25 mM HEPES buffer.

2. The pharmaceutical composition for promoting the proliferation of hair follicle-derived mesenchymal stem cells according to claim 1, characterized in that: The pharmaceutical composition consists of the following components: 10 ng / mL transforming growth factor β, 0.04 μM triptolide, 10 ng / mL vascular endothelial growth factor, 0.5 μg / mL grape seed extract, 1 mg / mL trace element solution, 1% hyaluronic acid, 0.1 μM cyclosporine, and 20 mM HEPES buffer.

3. The pharmaceutical composition for promoting the proliferation of hair follicle-derived mesenchymal stem cells according to claim 1, characterized in that: The trace element solution contains 0.5 μg / ml CuSO4, 0.16 μg / ml MnSO4, 0.3 μg / ml ZnSO4, 2.2 μg / ml CoCl2 6H2O, 0.45 μg / ml FeSO4, and the balance is water.

4. The method for preparing the pharmaceutical composition for promoting the proliferation of hair follicle-derived mesenchymal stem cells according to any one of claims 1 to 3, characterized in that: Transforming growth factor β, triptolide, vascular endothelial growth factor, grape seed extract, trace element solution, hyaluronic acid, cyclosporine and HEPES buffer are dissolved in DMEM / F12 and mixed to obtain the pharmaceutical composition for promoting the proliferation of hair follicle-derived mesenchymal stem cells.

5. A method for promoting the proliferation of hair follicle-derived mesenchymal stem cells, characterized in that: The method is for non-therapeutic use, comprising: culturing hair follicle-derived mesenchymal stem cells using the pharmaceutical composition according to any one of claims 1 to 3.

6. The method for promoting the proliferation of hair follicle-derived mesenchymal stem cells according to claim 5, characterized in that: The method comprises: (1) Cultivating hair follicle tissue, subculturing cells when they reach 80%-90% confluence, and then obtaining hair follicle-derived mesenchymal stem cells through digestion, pipetting, inoculation, culture observation, and identification; (2) When the hair follicle-derived mesenchymal stem cells reach the 3rd to 5th generation and the cells reach a confluence of 80% to 90%, the drug composition is added and cultured in a cell culture incubator at 37° C., 1% O 2 , 5% CO 2 , and 94% N 2 for 72 hours.

7. The method for promoting the proliferation of hair follicle-derived mesenchymal stem cells according to claim 6, characterized in that: In step (1), the culture and subculture conditions are: 37°C, 5% CO2 and 95% air, and the culture medium is DMEM / F12 containing 10v / v% fetal bovine serum.

8. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a product for preventing and treating hair loss.

9. Use of the method according to any one of claims 5 to 7 in preparing a product for preventing and treating hair loss.

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