Pharmaceutical composition for improving regeneration of cell therapy and application of pharmaceutical composition

Abstract

The embodiment of the invention discloses a pharmaceutical composition for improving cell therapy regeneration and application thereof. The pharmaceutical composition comprises 5 to 15 ng / mL of a transforming growth factor beta, 0.02 to 0.06 [mu] M of triptolide, 8 to 12 ng / mL of a vascular endothelial growth factor, 0.1 to 1 [mu] g / mL of a grape seed extract, 0.8 to 1.6 mg / mL of a trace element solution, 0.5 to 2% of hyaluronic acid, 0.05 to 0.15 [mu] M of cyclosporin and 15 to 25 mM of an HEPES buffer solution. The pharmaceutical composition provided by the invention can effectively promote proliferation and differentiation of hair follicle-derived mesenchymal stem cells, plays a better role in treating alopecia, has the advantages of small side effect, high safety, lower cost and wide applicable crowd, and has better application prospects in clinic.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of biomedicine technology, and specifically to a pharmaceutical composition for improving the regeneration of cell therapy 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 therapy such as minoxidil and finasteride, although commonly used to treat male pattern baldness, can promote hair growth or slow hair loss, but minoxidil needs to be used for a long time, and the effect may disappear after stopping the drug; although finasteride is effective, it may cause side effects. The combined use of these two drugs may enhance the therapeutic effect, but the applicable population is usually limited to patients with early hair loss. Hair transplant surgery, such as hair follicle transplantation, can restore hair growth, with obvious and lasting effects, but the cost is high and the operation is complicated. It is suitable for patients with large areas of hair loss or poor drug treatment effects. Other auxiliary treatment methods such as laser treatment, scalp massage and herbal treatment, although each has its own advantages, such as laser treatment is non-invasive but the effect varies from person to person, scalp massage is simple and easy but the effect is limited, herbal treatment is natural and has no side effects but the effect needs long-term observation.

[0003] Cell therapy uses the cells' own regenerative abilities to promote hair growth rather than just inhibit the hair loss process. 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 may create dependency, cell therapy has the potential to provide a long-term solution because it is based on the cells' own function and regenerative abilities. Cell therapy can be personalized to the patient's specific circumstances to improve the effectiveness of the treatment. Because cell therapy uses the patient's own or allogeneic cells, it has fewer side effects than some traditional therapies. But the success of cell therapy ultimately depends on the number of cells that reach the target tissue, their vitality, and their promotion of tissue regeneration. This key factor determines the effectiveness and durability of cell therapy in regenerative medicine.

[0004] A sufficient number of cells ensures that there are enough cells to settle in the hair follicles and start growing new hair. If the number of cells is insufficient, it may not cover all damaged hair follicle areas, thus affecting 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 the recovery of hair follicles and hair growth. By secreting growth factors and extracellular matrix components, cells can activate and promote the regeneration process of hair follicles. These factors can stimulate hair follicles to enter the growth phase from the resting phase, thereby promoting hair growth.

[0005] Therefore, when cell therapy is used to treat hair loss, the number of cells, their vitality, and their promotion of tissue regeneration are key factors in ensuring the effectiveness of the 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, an embodiment of the present invention provides a pharmaceutical composition for improving regeneration in cell therapy and its application.

[0007] In order to achieve the above purpose, the embodiment of the present invention provides 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 an important 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 ability of hair follicle-derived mesenchymal stem cells through the promotion of TGF-β, thereby accelerating the growth of new hair and improving the therapeutic effect. The transforming growth factor β used in the embodiment of the present invention comes from Okey Biotech (Cat. No.: P1230).

[0010] 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.

[0011] Vascular endothelial growth factor (VEGF) (CAS No.: 127464-60-2): also known as vascular permeability factor; angiostatic factor (VAS) or vascular regulator. It is a specific growth factor related to vascular growth purified from the culture fluid of pituitary stellate follicle cells and ovarian cells of various rodents, and has the effect of promoting vascular permeability. VEGF can bind to specific receptors (Flt-1, Flk-1 / KDR) on the surface of endothelial cells, promote the proliferation of vascular endothelial cells, and stimulate the formation of new blood vessels in the body. In addition, VEGF, as a local endogenous regulator, also 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, which have strong antioxidant capacity. They can remove free radicals in the body, reduce oxidative damage to cells, and thus protect cardiovascular and cerebrovascular systems. The grape seed extract used in the present embodiment comes from Shaanxi Panier Biotechnology Co., Ltd. (Article No.: PB20211217).

[0013] Trace elements play an important role in hair loss, and they play a vital role in hair growth and health. The role of trace elements in hair loss is mainly to maintain the growth environment of hair, promote healthy hair growth and prevent hair loss. Maintaining appropriate trace element levels is essential for preventing and treating hair loss. The trace element solution used in the embodiment of the present invention comes from Shanghai Huzhen Industrial Co., Ltd. (Article No.: HZ1941-50L), and the composition is: 50μg CuSO4, 16μg MnSO4, 30μg ZnSO4, 220μg CoCl2 6H2O, 45μg FeSO4, add water to 100ml.

[0014] Hyaluronic acid (CAS No.: 9004-61-9): It is the main component of connective tissues such as the intercellular matrix, vitreous body, and synovial fluid of the human body. It plays an important physiological function in the body, including water retention, maintaining extracellular space, regulating osmotic pressure, lubrication, and promoting cell repair. As the main component of the intercellular matrix, hyaluronic acid is directly involved in the regulation of electrolyte exchanges inside and outside cells, and plays a role as a filter for physical and molecular information. The hyaluronic acid used in the embodiment of the present invention comes from Tonghui Pharmaceutical.

[0015] Cyclosporine (CAS No. 59865-13-3): It is an immunosuppressant that is often used to prevent rejection after organ transplantation and can also be used to treat some autoimmune diseases. The purpose of its use is to reduce rejection and help the newly implanted hair follicles grow stably.

[0016] HEPES buffer (CAS No. 7365-45-9): The most suitable pH value for the head is usually between 4.5 and 5.5. This range of pH helps maintain the scalp's natural protective barrier and keeps the scalp healthy. The acidic environment of the scalp helps prevent the growth of bacteria and fungi, reducing the occurrence of 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 regeneration in cell therapy 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 regeneration in cell therapy.

[0019] According to a third aspect of an embodiment 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] Further, the method comprises:

[0021] (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;

[0022] (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.

[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 the preparation of a product for preventing and treating hair loss.

[0025] According to a fifth aspect of an embodiment 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 treatment methods;

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

[0031] To 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] 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.

[0033] Figure 1 The cell proliferation results of the hair follicle-derived mesenchymal stem cells provided by the present invention when cultured to the 7th day and the 11th day;

[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 provided by the present invention under different induction environments;

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

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

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

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

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

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

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

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

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

[0045] 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.

[0046] 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.

[0047] Example 1

[0048] The present embodiment provides a pharmaceutical composition for improving regeneration in cell therapy, which is composed of: 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, 20 mM HEPES buffer, with DMEM / F12 as solvent.

[0049] Example 2

[0050] The present embodiment provides a pharmaceutical composition for improving regeneration in cell therapy, which is composed of: 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, 16 mM HEPES buffer, with DMEM / F12 as solvent.

[0051] Example 3

[0052] The present embodiment provides a pharmaceutical composition for improving regeneration in cell therapy, which is composed of: 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, 22 mM HEPES buffer, with DMEM / F12 as solvent.

[0053] Comparative Example 1

[0054] The present comparative example provides a pharmaceutical composition for improving the regeneration of cell therapy, which is composed of: 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, 20 mM HEPES buffer, with DMEM / F12 as solvent.

[0055] Comparative Example 2

[0056] The comparative example provides a pharmaceutical composition for improving the regeneration of 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, 20 mM HEPES buffer, and DMEM / F12 as solvent.

[0057] Comparative Example 3

[0058] This comparative example provides a pharmaceutical composition for improving the regeneration of 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, 16 mM HEPES buffer, with DMEM / F12 as solvent.

[0059] Example 4

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

[0061] (1) Obtaining 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, 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.

[0063] In vitro culture: Cut the extracted hair follicle tissue into small pieces, add them evenly to the well plate with complete culture medium, and continue to culture in a cell culture incubator at 37°C and 5% CO2. After 48 hours, replace half of the medium, and continue to observe the migration of stem cells from the tissue block after 7-10 days.

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

[0065] Digest cells: Aspirate the culture medium in the culture flask and wash the cells once with PBS. Then add an appropriate amount of trypsin digestion solution (such as 0.25% trypsin) and warm it at 37℃ for about 3 minutes. Observe under an inverted microscope. After the cells shrink and become round, aspirate the digestion solution and add complete culture solution to stop digestion.

[0066] 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 15ml of fresh complete culture medium and continue to culture in a cell culture incubator at 37℃ and 5% CO2.

[0067] 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.

[0068] 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.

[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 are 80%-90% grown.

[0070] (2) Cell culture: The culture medium supernatant in the culture dish was aspirated, and the dish was 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 then 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 The cell proliferation of hair follicle-derived mesenchymal stem cells isolated and extracted from hair follicle tissue observed under a microscope (magnification 4X) at the 7th and 11th days of culture was presented. The culture conditions were 37°C, 5% CO2 and 95% air, with DMEM / F12 containing 10v / v% fetal bovine serum as the culture medium. The results showed that the cells were polygonal or star-shaped, with relatively small cell size and dense arrangement.

[0074] 2. Survival of cells under different induction environments

[0075] Figure 2 Figures A, H 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 the 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, the 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). Cell apoptosis and necrosis were also observed in experimental groups 1-3 and control groups 1-3, but the proportion of cell necrosis and apoptosis in experimental group 1 was significantly lower than that in other groups, and the integrity of its cell membrane was significantly better than that in other groups. This result shows that the culture conditions of experimental group 1 provide a more favorable living environment, thereby effectively slowing down the process of cell 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 carried out for 6 hours at 37°C and 5% carbon dioxide concentration. After the cells completed adherent growth, the culture conditions of each group were changed, wherein 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). At 12 hours, 1 day, 3 days, 5 days, and 7 days, a cell counter was used to measure the number of cells in each subgroup. The results are shown in Tables 1 and Figure 3 .

[0088] Table 1

[0089]

[0090] The experimental results showed that within 12 hours to 3 days after the cells adhered, the number of cells in experimental group 1 showed an increasing trend, reaching 1.86 times before inoculation, but did not reach exponential growth. From the 3rd day, cells began to undergo apoptosis and necrosis, and by the 7th day, the number of cells dropped to 1.1 times before inoculation. This result shows that the induction environment of experimental group 1 can promote the good growth ability of cells in the early stage. Although the number of surviving cells decreased over time, it was still not lower than the number of cells before inoculation, indicating that the induction environment can maintain cell survival under low oxygen and low nutrition conditions. In contrast, the number of cells in the control group continued to decrease within 12 hours to 7 days after the cells adhered, and by the 7th day it dropped to 1 / 15 of the number before inoculation. The number of cells in experimental groups 2 and 3 remained basically unchanged within 12 hours to 3 days after the cells adhered, but from the 3rd day, the cells continued to undergo apoptosis and necrosis, and by the 7th day, the number of cells in experimental group 2 dropped to 5 / 6 of the number before inoculation, while the number of cells in experimental group 3 dropped to 1 / 3 of the number before inoculation. The number of cells in control group 1 remained basically unchanged from 12 hours to 1 day after cell attachment, but from the first day onwards, cells continued to undergo apoptosis and necrosis, and by the seventh day, the number of cells in control group 1 had dropped to 3 / 20 of that before inoculation. The number of cells in control group 2 and control group 3 continued to drop from 12 hours to 7 days after cell attachment, and by the seventh day, the number of cells in control group 2 had dropped to 1 / 10 of that before inoculation, and the number of cells in control group 3 had 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 allocated 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 bottle, and an appropriate amount of liquid was added to the culture bottle to ensure that the six-well plate adhered to the wall. Incubate in a culture box for 2-4 hours to allow the cells to adhere to the wall. 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 live cells, and the rate of agglomeration in each well were counted and analyzed. The results are shown in Table 2 below.

[0093] Table 2

[0094]

[0095] The 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 agglomeration rate was maintained at the lowest level. In contrast, the number of live cells in other groups decreased significantly, and the cell agglomeration rate increased significantly. Specifically, the cell agglomeration rates of the control group, experimental group 3, and control groups 1-2 all increased to more than 50%, among which the cell agglomeration 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 agglomeration 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, with a total of 3 treatments. The efficacy evaluation was analyzed using head hair images. The experimental results showed that the number of hair growing in the treatment area of ​​the 8 patients increased significantly, and the proportion of growing hair increased (such as Figure 4-Figure 11 As shown, the left picture is before treatment, and the right picture is after treatment). In addition, after 3 treatments, the amount of hair growth of a hair loss patient increased, but the effect was not very obvious (such as Fig.12 , the left picture is before treatment, 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 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. A pharmaceutical composition for improving regeneration in cell therapy, characterized in that: The pharmaceutical composition comprises: 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 improving cell therapy regeneration according to claim 1, characterized in that: The pharmaceutical composition comprises: 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 improving cell therapy regeneration 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 improving cell therapy regeneration 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 improving cell therapy regeneration.

5. A method for improving regeneration in cell therapy, characterized in that The method comprises: culturing hair follicle-derived mesenchymal stem cells using the pharmaceutical composition according to any one of claims 1 to 3.

6. The method for improving regeneration in cell therapy 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 improving regeneration in cell therapy 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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