Mesenchymal stem cell hair growth liquid and preparation method thereof

Abstract

The invention relates to the technical field of biological products, and discloses a mesenchymal stem cell hair growth liquid and a preparation method thereof, and the mesenchymal stem cell hair growth liquid comprises the following components: hair follicle mesenchymal stem cells; a hair follicle mesenchymal stem cell exosome; growth factors: a vascular endothelial growth factor (VEGF), a fibroblast growth factor (FGFs), a keratinocyte growth factor (KGF-7) and interleukin-10 (IL-10); vitamins: vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C and biotin; the plant extracts comprise a cacumen biotae extract, a polygonum multiflorum extract, a ginseng extract, a peppermint extract, a purslane extract and a rhodiola rosea extract; other active ingredients: tripeptide and digoxin; the auxiliary material is a hyaluronic acid-collagen peptide graft copolymer; and the balance of a phosphate buffer solution.

Description

Technical Field

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

[0002] Hair loss is a common health concern that seriously impacts patients' quality of life and mental well-being. The causes of hair loss vary, including genetic factors, endocrine disorders (such as androgenic alopecia, primarily caused by the adverse effects of androgens and their metabolite dihydrotestosterone on hair follicles), malnutrition, stress, and side effects of certain diseases or medications. Currently, numerous methods and products are available on the market for treating hair loss, but all have limitations. In terms of drug treatment, minoxidil is a commonly used topical medication that requires long-term continuous use, and some patients may experience adverse reactions such as skin irritation, itching, and redness. Finasteride, an oral medication, although effective for androgenic alopecia, may cause serious side effects such as sexual dysfunction and is not suitable for women and children. Hair transplant surgery carries risks such as high trauma, high cost, unstable hair follicle survival rate, and the possibility of infection. Traditional hair growth products, such as some plant extract shampoos or serums, often have difficulty achieving significant hair growth effects due to low levels of active ingredients and unclear mechanisms of action. Mesenchymal stem cells (MSCs) have recently garnered significant attention in the field of regenerative medicine due to their multipotential differentiation, immunomodulatory functions, and ability to secrete diverse bioactive factors. They demonstrate significant potential in hair regeneration, particularly in hair follicle regeneration. Studies have shown that the growth factors and cytokines secreted by MSCs can modulate the biological behavior of hair follicle cells, promoting the activation, proliferation, and differentiation of hair follicle stem cells and improving the follicle microenvironment, thus creating favorable conditions for hair regeneration. However, the efficient utilization of MSCs, the secretion of sufficient concentrations and diversity of hair-promoting factors, and the stable application of these factors in hair growth products remain pressing challenges. Summary of the Invention

[0003] (1) Technical problems solved In view of the deficiencies in the prior art, the present invention provides a mesenchymal stem cell hair growth fluid and a preparation method thereof.

[0004] (2) Technical solution To achieve the above objectives, the present invention provides the following technical solution: a mesenchymal stem cell hair growth solution, comprising the following components: Hair follicle mesenchymal stem cells 2~5%; Hair follicle mesenchymal stem cell exosomes 0.02~0.05%; Growth factors: vascular endothelial growth factor (VEGF) 0.002~0.005%, fibroblast growth factors (FGFs) 0.003~0.005%, keratinocyte growth factor (KGF-7) 0.001~0.002%, interleukin-10 (IL-10) 0.001~0.002%; Vitamins: Vitamin E 0.001~0.002%, Vitamin B5 0.0008~0.001%, Vitamin B2 0.0006~0.0009%, Vitamin B6 0.0002~0.0005%, Vitamin C 0.001~0.002%, Biotin 0.007~0.009%; Plant extracts: Platycladus orientalis leaf extract 1-3%, Polygonum multiflorum extract 2-4%, Ginseng extract 0.5-2%, Peppermint extract 0.5-2%, Portulaca oleracea extract 0.8-1.5%, Rhodiola rosea extract 0.6-0.8%; Other active ingredients: Tripeptide 0.00015~0.0002%, Digoxin 0.0001~0.0003%; Excipients: hyaluronic acid-collagen peptide graft copolymer 5~8%; Balance: phosphate buffer solution.

[0005] Furthermore, the method for preparing hair follicle mesenchymal stem cells comprises the following steps: A1, basal medium: DMEM / F12 medium containing L-glutamine and 15 mM HEPES; Supplementary factors: fetal bovine serum (final concentration 5%), 100 U / mL penicillin-streptomycin mixture, 10 ng / mL epidermal growth factor, 5 ng / mL fibroblast growth factor-2, and 50 μg / mL ascorbic acid; Digestion solution: 0.25% trypsin-EDTA; Buffer: PBS, pH 7.2–7.4; A2. Take scalp tissue, rinse with PBS three times to remove blood stains, and cut into 1 mm 3 Separate intact hair follicles from small pieces and soak them in PBS containing antibiotics for 10 minutes to sterilize. Use sterile scissors to cut off the upper hair shaft of the hair follicle, retaining the hair bulb containing the dermal papilla and the lower sheath. Transfer the treated hair follicles to 0.25% trypsin-EDTA and digest them at 37°C for 20 minutes. During this period, gently pipette to disperse the cells. Add medium containing 5% FBS to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in basal medium. A3. Adjust the cell density to 5×10 4Cells were seeded into gelatin-coated 60 mm culture dishes with 0.1% gelatin and incubated at 37°C for 1 hour. The culture medium was not changed within 48 hours, and fresh culture medium was replaced every 3 days thereafter. Cells were passaged when the confluence reached 70-80%. A4. Aspirate and discard the old culture medium, rinse twice with PBS, add 2 mL of trypsin-EDTA, and incubate at 37°C for 3–5 min. Add an equal amount of serum-containing culture medium to terminate digestion. Gently pipette the bottom of the culture dish to dislodge the cells. Collect the cell suspension and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the pellet in fresh culture medium, and inoculate it onto a new gelatin-coated culture dish at a 1:3 ratio. When the confluence of A5 and passage 3 cells reached 50%, they were transferred to a hypoxic incubator with an oxygen concentration of 5% and cultured for 48 h. Wnt-3a was added to the culture medium at a concentration of 20 ng / mL and cultured for 3 consecutive days. A collagen-hyaluronic acid scaffold was used with a concentration of 0.5% and the cells were seeded into the scaffold to form a three-dimensional spheroid culture. A6. Take cells in logarithmic growth phase, digest them and centrifuge them, resuspend the pellet in freezing buffer containing 90% FBS + 10% DMSO, and adjust the density to 1×10 6 / mL, dispense into cryopreservation tubes, store in liquid nitrogen for a long time, and thaw after the cryopreservation tubes are taken out of liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000 rpm for 5 min, discard the cryopreservation solution, resuspend in fresh culture medium and inoculate, and change the medium after 24 h.

[0006] Furthermore, the hair follicle mesenchymal stem cells are P3~P5 generation cells, and the cell activity is ≥95%.

[0007] Furthermore, the method for preparing hair follicle mesenchymal stem cell exosomes comprises the following steps: B1. Take P3-P5 hair follicle mesenchymal stem cells and culture them in a T75 culture flask using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell confluence reaches 70-80%, discard the original medium and gently rinse the cells 2-3 times with PBS buffer to remove residual serum. Then replace the medium with serum-free medium and continue to culture in a CO2 incubator for 48-72 hours. B2. Collect the culture supernatant and transfer it to a centrifuge tube. Centrifuge at 300 g for 10 min at 4°C. Then transfer the supernatant to a new centrifuge tube and centrifuge at 2000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane, collect the filtrate, transfer the filtrate to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4000 g for 30-60 min at 4°C until the volume of the concentrate is 1 / 10-1 / 20 of the original volume. Collect the concentrate. B3. Prepare sucrose gradient solution. Add 60%, 40%, 30%, and 20% sucrose solution to the ultracentrifuge tube in sequence. The volume of each layer of solution is equal. Add gently to avoid disrupting the gradient. Slowly layer the ultrafiltration concentrated sample on the upper layer of the sucrose gradient solution. Ultracentrifuge at 100,000g for 16 hours at 4°C. After centrifugation, use a syringe to collect fractions of different densities from the bottom of the centrifuge tube in sequence, and collect the fraction containing exosomes. B4. Dilute the exosome-containing fraction with PBS buffer and transfer it to a 30 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 g for 30 min at 4°C. Discard the filtrate. Repeat this step 2-3 times and collect the concentrated solution in the ultrafiltration centrifuge tube, which is the purified hair follicle mesenchymal stem cell exosomes.

[0008] Furthermore, the fibroblast growth factors (FGFs) include FGF-2 and FGF-7, with a mass ratio of 2:1.

[0009] Furthermore, the preparation method of the plant extract comprises the following steps: Platycladus orientalis leaf extract: Take dried Platycladus orientalis leaves, grind them, and soak them in 70-80% ethanol solution at a solid-liquid ratio of 1g:8-10mL for 24-48 hours. Ultrasonic extraction is performed for 30-60 minutes. The filtrate is filtered and concentrated under reduced pressure until there is no alcohol taste to obtain the Platycladus orientalis leaf extract. Polygonum multiflorum extract: After cutting Polygonum multiflorum, reflux extract it with 50-60% ethanol solution at a material-liquid ratio of 1 g: 10-12 mL for 2-3 times, each time for 1-2 hours. Combine the extracts and concentrate under reduced pressure to obtain the Polygonum multiflorum extract; Ginseng extract: After crushing ginseng, soak it in 70-80% ethanol solution at a material-liquid ratio of 1g:12-15mL for 36-48h, extract by percolation, collect the percolation liquid, and concentrate under reduced pressure to obtain the ginseng extract; Peppermint extract: Peppermint is dried and crushed, and soaked in a 40-50% ethanol solution at a solid-liquid ratio of 1 g:6-8 mL for 24-36 hours. Reflux extraction is performed for 1-2 hours, filtered, and the filtrate is concentrated under reduced pressure to obtain the peppermint extract. Purslane extract: Purslane is dried and crushed, and reflux-extracted with 70-80% ethanol solution at a solid-liquid ratio of 1 g:8-10 mL for 2-3 times, each time for 1.5-2.5 hours. The extracts are combined and concentrated under reduced pressure to obtain the purslane extract; Rhodiola rosea extract: After the rhizome of Rhodiola rosea is sliced, it is refluxed and extracted 2 to 3 times with 60-70% ethanol solution at a solid-liquid ratio of 1 g:10-15 mL, each time for 2 to 3 hours. The extracts are combined and concentrated under reduced pressure to obtain the Rhodiola rosea extract.

[0010] Furthermore, the preparation method of the hyaluronic acid-collagen peptide graft copolymer comprises the following steps: C1. Dissolve 1-3 parts of hyaluronic acid in 50-100 parts of PBS and stir magnetically for 10-30 minutes to obtain a hyaluronic acid solution. Add 0.3-0.5 parts of EDC and 0.15-0.2 parts of NHS and activate at 30°C with constant stirring for 1-1.5 hours. C2. Dissolve 0.5-1 part collagen peptide in 50-80 parts deionized water. After complete dissolution, slowly add it dropwise to the above hyaluronic acid solution. React at 35°C under nitrogen protection for 6-8 hours. Maintain magnetic stirring at 100-200 rpm during this period. C3. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed with deionized water for 48-72 hours, with the deionized water replaced every 6 hours. The dialyzed graft copolymer solution is placed in a freeze dryer and freeze-dried at -50°C and 0.01 mbar for 24 hours to obtain hyaluronic acid-collagen peptide graft copolymer powder, which is then sealed and stored for later use. C4. Take 0.5-1 parts of freeze-dried graft copolymer powder and dissolve it in 50-100 parts of deionized water to prepare a solution. Then use high-pressure homogenization method: 10 cycles of homogenization at 800 bar pressure to form a nanoparticle suspension with a particle size of 100-120 nm and a particle size distribution (PDI) ≤ 0.15.

[0011] Furthermore, the tripeptide is a copper peptide (GHK-Cu), wherein the molar ratio of copper ions to the tripeptide is 1:1.

[0012] Furthermore, a method for preparing a mesenchymal stem cell hair growth solution comprises the following steps: S1. Take the formulated amount of hyaluronic acid-collagen peptide graft copolymer, add 2 / 3 phosphate buffer solution, and stir on a stirrer until completely dissolved to form an excipient solution; S2. Add Platycladus orientalis leaf extract, Polygonum multiflorum extract, Panax ginseng extract, Mentha piperita extract, Portulaca oleracea extract, and Rhodiola rosea extract to the above-mentioned auxiliary material solution in proportion, and stir at medium speed for 10 to 30 minutes; S3. Add vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C, and biotin to the above mixture in proportion, and continue stirring for 10-20 minutes; S4. Add vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, interleukin-10, tripeptide, and digoxin in proportion and stir for 10-15 minutes; S5. Under a sterile environment, slowly add hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes into the above mixture in proportion and gently stir for 5-10 minutes; S6. Adjust the pH value of the mixture to 6.8-7.2 with phosphate buffer solution, add the remaining phosphate buffer solution to make the mixture reach the specified volume, stir evenly, filter the mixture through a 0.22 μm filter membrane to sterilize, and obtain the final hair follicle mesenchymal stem cell hair growth fluid. Under sterile conditions, divide the hair growth fluid into sterile containers, seal them, and store them at 2-8°C.

[0013] (3) Beneficial technical effects The present invention utilizes hair follicle mesenchymal stem cells and exosomes in synergy with growth factors, plant extracts, and other ingredients to promote hair growth through multiple mechanisms, including repairing hair follicles, regulating the growth cycle, and optimizing the scalp microenvironment, effectively enhancing hair growth. Furthermore, the use of a hyaluronic acid-collagen peptide graft copolymer nanocarrier provides strong targeting and improves the utilization rate of the active ingredient. The product is highly safe, reduces the incidence of skin irritation, and has no systemic adverse reactions, making it suitable for long-term use. The process is stable, and optimized cultivation and preparation processes ensure the stability of the active ingredients, providing an efficient, safe, and stable solution for hair loss treatment. DETAILED DESCRIPTION

[0014] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only 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 making any creative efforts shall fall within the scope of protection of the present invention.

[0015] The components of the mesenchymal stem cell hair growth solution of the present invention were all purchased from the market unless otherwise specified. The raw materials in the present invention are measured by mass volume percentage (w / v%); The parts used in the present invention are all weight fractions; Example 1 A mesenchymal stem cell hair growth solution, comprising the following components: Hair follicle mesenchymal stem cells 2%; Hair follicle mesenchymal stem cell exosomes 0.02%; Growth factors: vascular endothelial growth factor (VEGF) 0.002%, fibroblast growth factors (FGFs) 0.003%, keratinocyte growth factor (KGF-7) 0.001%, interleukin-10 (IL-10) 0.001%; Vitamins: Vitamin E 0.001%, Vitamin B5 0.0008%, Vitamin B2 0.0006%, Vitamin B6 0.0002%, Vitamin C 0.001%, Biotin 0.007%; Plant extracts: Platycladus orientalis leaf extract 1%, Polygonum multiflorum extract 2%, Ginseng extract 0.5%, Mentha piperita extract 0.5%, Portulaca oleracea extract 0.8%, Rhodiola rosea extract 0.6%; Other active ingredients: Tripeptide 0.00015%, Digoxin 0.0001%; Excipients: hyaluronic acid-collagen peptide graft copolymer 5%; Balance: phosphate buffer solution.

[0016] Hair follicle mesenchymal stem cells are P3 cells with cell activity ≥95%.

[0017] Fibroblast growth factors (FGFs) include FGF-2 and FGF-7, with a mass ratio of 2:1.

[0018] The method for preparing hair follicle mesenchymal stem cells comprises the following steps: A1, basal medium: DMEM / F12 medium containing L-glutamine and 15 mM HEPES; Supplementary factors: fetal bovine serum (final concentration 5%), 100 U / mL penicillin-streptomycin mixture, 10 ng / mL epidermal growth factor, 5 ng / mL fibroblast growth factor-2, and 50 μg / mL ascorbic acid; Digestion solution: 0.25% trypsin-EDTA; Buffer: PBS, pH 7.2; A2. Take scalp tissue, rinse with PBS three times to remove blood stains, and cut into 1 mm 3 Separate intact hair follicles from small pieces and soak them in PBS containing antibiotics for 10 minutes to sterilize. Use sterile scissors to cut off the upper hair shaft of the hair follicle, retaining the hair bulb containing the dermal papilla and the lower sheath. Transfer the treated hair follicles to 0.25% trypsin-EDTA and digest them at 37°C for 20 minutes. During this period, gently pipette to disperse the cells. Add medium containing 5% FBS to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in basal medium. A3. Adjust the cell density to 5×10 4 Cells were seeded into gelatin-coated 60 mm culture dishes with 0.1% gelatin and incubated at 37°C for 1 hour. The culture medium was not changed within 48 hours, and fresh culture medium was replaced every 3 days thereafter. Cells were passaged when the confluence reached 70-80%. A4. Aspirate and discard the old culture medium, rinse twice with PBS, add 2 mL of trypsin-EDTA, incubate at 37°C for 3 min, add an equal amount of serum-containing culture medium to terminate digestion, gently pipette the bottom of the culture dish to detach the cells, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in fresh culture medium, and inoculate it onto a new gelatin-coated culture dish at a 1:3 ratio; When the confluence of A5 and passage 3 cells reached 50%, they were transferred to a hypoxic incubator with an oxygen concentration of 5% and cultured for 48 h. Wnt-3a was added to the culture medium at a concentration of 20 ng / mL and cultured for 3 consecutive days. A collagen-hyaluronic acid scaffold was used with a concentration of 0.5% and the cells were seeded into the scaffold to form a three-dimensional spheroid culture. A6. Take cells in logarithmic growth phase, digest them and centrifuge them, resuspend the pellet in freezing buffer containing 90% FBS + 10% DMSO, and adjust the density to 1×10 6 / mL, dispense into cryopreservation tubes, store in liquid nitrogen for a long time, and thaw after the cryopreservation tubes are taken out of liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000 rpm for 5 min, discard the cryopreservation solution, resuspend in fresh culture medium and inoculate, and change the medium after 24 h.

[0019] The method for preparing hair follicle mesenchymal stem cell exosomes comprises the following steps: B1. P3 hair follicle mesenchymal stem cells were cultured in a T75 culture flask using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell confluence reached 70-80%, the original medium was discarded and the cells were gently rinsed twice with PBS buffer to remove residual serum. The medium was then replaced with serum-free medium and cultured in a CO2 incubator for 48 h. B2. Collect the culture supernatant and transfer it to a centrifuge tube. Centrifuge at 300 g for 10 min at 4°C. Then transfer the supernatant to a new centrifuge tube and centrifuge at 2000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane, collect the filtrate, transfer the filtrate to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4000 g for 30 min at 4°C until the volume of the concentrate is 1 / 10 of the original volume. Collect the concentrate. B3. Prepare sucrose gradient solution. Add 60%, 40%, 30%, and 20% sucrose solution to the ultracentrifuge tube in sequence. The volume of each layer of solution is equal. Add gently to avoid disrupting the gradient. Slowly layer the ultrafiltration concentrated sample on the upper layer of the sucrose gradient solution. Ultracentrifuge at 100,000g for 16 hours at 4°C. After centrifugation, use a syringe to collect fractions of different densities from the bottom of the centrifuge tube in sequence, and collect the fraction containing exosomes. B4. Dilute the exosome-containing fraction with PBS buffer and transfer it to a 30 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 g for 30 min at 4°C. Discard the filtrate. Repeat this step twice and collect the concentrate in the ultrafiltration centrifuge tube, which is the purified hair follicle mesenchymal stem cell exosomes.

[0020] The preparation method of the plant extract comprises the following steps: Platycladus orientalis leaf extract: Dried Platycladus orientalis leaves were ground and soaked in 70% ethanol solution at a solid-liquid ratio of 1g:8mL for 24h. Ultrasonic extraction was performed for 30min. The filtrate was filtered and concentrated under reduced pressure until there was no alcohol smell to obtain the Platycladus orientalis leaf extract. Polygonum multiflorum extract: After cutting Polygonum multiflorum, reflux extract with 50% ethanol solution at a solid-liquid ratio of 1g:10mL twice, each time for 1 hour, combine the extracts, and concentrate under reduced pressure to obtain Polygonum multiflorum extract; Ginseng extract: Ginseng was crushed and soaked in 70% ethanol solution at a solid-liquid ratio of 1 g:12 mL for 36 h. The percolation was performed and the percolation liquid was collected and concentrated under reduced pressure to obtain the ginseng extract. Peppermint extract: Peppermint was dried and crushed, soaked in 40% ethanol solution at a solid-liquid ratio of 1 g:6 mL for 24 h, refluxed for 1 h, filtered, and the filtrate was concentrated under reduced pressure to obtain the peppermint extract; Purslane extract: Purslane was dried and ground, and refluxed with 70% ethanol solution at a solid-liquid ratio of 1 g:8 mL twice, each time for 1.5 h. The extracts were combined and concentrated under reduced pressure to obtain the purslane extract; Rhodiola rosea extract: After the rhizome of Rhodiola rosea was sliced, it was reflux extracted twice with 60% ethanol solution at a solid-liquid ratio of 1g:10mL, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain the Rhodiola rosea extract.

[0021] The preparation method of the hyaluronic acid-collagen peptide graft copolymer comprises the following steps: C1. Dissolve 1 part of hyaluronic acid in 50 parts of PBS and stir magnetically for 10 minutes to obtain a hyaluronic acid solution. Add 0.3 parts of EDC and 0.15 parts of NHS and activate at 30°C with constant stirring for 1 hour. C2. Dissolve 0.5 parts of collagen peptide in 50 parts of deionized water. After complete dissolution, slowly add it dropwise to the above hyaluronic acid solution. React at 35°C under nitrogen protection for 6 hours with magnetic stirring at 100 rpm. C3. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed with deionized water for 48 hours, with the deionized water replaced every 6 hours. The dialyzed graft copolymer solution is placed in a freeze dryer and freeze-dried at -50°C and 0.01 mbar for 24 hours to obtain hyaluronic acid-collagen peptide graft copolymer powder, which is sealed and stored for later use; C4. Take 0.5 parts of the freeze-dried graft copolymer powder and dissolve it in 50 parts of deionized water to prepare a solution. Then use high-pressure homogenization method: 10 cycles of homogenization at a pressure of 800 bar to form a nanoparticle suspension with a particle size of 100-120 nm and a particle size distribution (PDI) ≤ 0.15.

[0022] The tripeptide is a copper peptide (GHK-Cu), in which the molar ratio of copper ion to tripeptide is 1:1.

[0023] A method for preparing a mesenchymal stem cell hair growth solution comprises the following steps: S1. Take the formulated amount of hyaluronic acid-collagen peptide graft copolymer, add 2 / 3 phosphate buffer solution, and stir on a stirrer until completely dissolved to form an excipient solution; S2. Add Platycladus orientalis leaf extract, Polygonum multiflorum extract, Panax ginseng extract, Mentha piperita extract, Portulaca oleracea extract, and Rhodiola rosea extract to the above-mentioned auxiliary material solution in proportion, and stir at medium speed for 10 minutes; S3. Add vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C, and biotin to the above mixture in proportion, and continue stirring for 10 minutes; S4, sequentially add vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, interleukin-10, tripeptide, and digoxin in proportion, and stir for 10 minutes; S5. Under a sterile environment, slowly add hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes into the above mixture in proportion and gently stir for 5 minutes; S6. Adjust the pH value of the mixture to 6.8 with phosphate buffer solution, add the remaining phosphate buffer solution to make the mixture reach the specified volume, stir evenly, filter the mixture through a 0.22 μm filter membrane to sterilize, and obtain the final hair follicle mesenchymal stem cell hair growth fluid. Under sterile conditions, divide the hair growth fluid into sterile containers, seal them, and store them at 2°C.

[0024] Example 2 A mesenchymal stem cell hair growth solution, comprising the following components: Hair follicle mesenchymal stem cells 4%; Hair follicle mesenchymal stem cell exosomes 0.03%; Growth factors: vascular endothelial growth factor (VEGF) 0.003%, fibroblast growth factors (FGFs) 0.004%, keratinocyte growth factor (KGF-7) 0.0015%, interleukin-10 (IL-10) 0.0015%; Vitamins: Vitamin E 0.0015%, Vitamin B5 0.0009%, Vitamin B2 0.0008%, Vitamin B6 0.0003%, Vitamin C 0.0015%, Biotin 0.008%; Plant extracts: Platycladus orientalis leaf extract 2%, Polygonum multiflorum extract 3%, Ginseng extract 0.8%, Mentha piperita extract 1.5%, Portulaca oleracea extract 1.2%, Rhodiola rosea extract 0.7%; Other active ingredients: Tripeptide 0.00018%, Digoxin 0.0002%; Excipients: hyaluronic acid-collagen peptide graft copolymer 6%; Balance: phosphate buffer solution.

[0025] Hair follicle mesenchymal stem cells are P4 cells with cell activity ≥95%.

[0026] Fibroblast growth factors (FGFs) include FGF-2 and FGF-7, with a mass ratio of 2:1.

[0027] The method for preparing hair follicle mesenchymal stem cells comprises the following steps: A1, basal medium: DMEM / F12 medium containing L-glutamine and 15 mM HEPES; Supplementary factors: fetal bovine serum (final concentration 5%), 100 U / mL penicillin-streptomycin mixture, 10 ng / mL epidermal growth factor, 5 ng / mL fibroblast growth factor-2, and 50 μg / mL ascorbic acid; Digestion solution: 0.25% trypsin-EDTA; Buffer: PBS, pH 7.2; A2. Take scalp tissue, rinse with PBS three times to remove blood stains, and cut into 1 mm 3 Separate intact hair follicles from small pieces and soak them in PBS containing antibiotics for 10 minutes to sterilize. Use sterile scissors to cut off the upper hair shaft of the hair follicle, retaining the hair bulb containing the dermal papilla and the lower sheath. Transfer the treated hair follicles to 0.25% trypsin-EDTA and digest them at 37°C for 20 minutes. During this period, gently pipette to disperse the cells. Add medium containing 5% FBS to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in basal medium. A3. Adjust the cell density to 5×10 4 Cells were seeded into gelatin-coated 60 mm culture dishes with 0.1% gelatin and incubated at 37°C for 1 hour. The culture medium was not changed within 48 hours, and fresh culture medium was replaced every 3 days thereafter. Cells were passaged when the confluence reached 70-80%. A4. Aspirate and discard the old culture medium, rinse twice with PBS, add 2 mL of trypsin-EDTA, incubate at 37°C for 4 min, add an equal amount of serum-containing culture medium to terminate digestion, gently pipette the bottom of the culture dish to detach the cells, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in fresh culture medium, and inoculate it onto a new gelatin-coated culture dish at a 1:3 ratio; When the confluence of A5 and passage 3 cells reached 50%, they were transferred to a hypoxic incubator with an oxygen concentration of 5% and cultured for 48 h. Wnt-3a was added to the culture medium at a concentration of 20 ng / mL and cultured for 3 consecutive days. A collagen-hyaluronic acid scaffold was used with a concentration of 0.5% and the cells were seeded into the scaffold to form a three-dimensional spheroid culture. A6. Take cells in logarithmic growth phase, digest them and centrifuge them, resuspend the pellet in freezing buffer containing 90% FBS + 10% DMSO, and adjust the density to 1×10 6 / mL, dispense into cryopreservation tubes, store in liquid nitrogen for a long time, and thaw after the cryopreservation tubes are taken out of liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000 rpm for 5 min, discard the cryopreservation solution, resuspend in fresh culture medium and inoculate, and change the medium after 24 h.

[0028] The method for preparing hair follicle mesenchymal stem cell exosomes comprises the following steps: B1. P4 hair follicle mesenchymal stem cells were cultured in a T75 culture flask using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell confluence reached 70-80%, the original medium was discarded and the cells were gently rinsed three times with PBS buffer to remove residual serum. The medium was then replaced with serum-free medium and cultured in a CO2 incubator for 50 h. B2. Collect the culture supernatant and transfer it to a centrifuge tube. Centrifuge at 300 g for 10 min at 4°C. Then transfer the supernatant to a new centrifuge tube and centrifuge at 2000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane, collect the filtrate, transfer the filtrate to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4000 g for 40 min at 4°C until the volume of the concentrate is 1 / 15 of the original volume. Collect the concentrate. B3. Prepare sucrose gradient solution. Add 60%, 40%, 30%, and 20% sucrose solution to the ultracentrifuge tube in sequence. The volume of each layer of solution is equal. Add gently to avoid disrupting the gradient. Slowly layer the ultrafiltration concentrated sample on the upper layer of the sucrose gradient solution. Ultracentrifuge at 100,000g for 16 hours at 4°C. After centrifugation, use a syringe to collect fractions of different densities from the bottom of the centrifuge tube in sequence, and collect the fraction containing exosomes. B4. Dilute the exosome-containing fraction with PBS buffer and transfer it to a 30 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 g for 30 min at 4°C. Discard the filtrate. Repeat this step twice and collect the concentrate in the ultrafiltration centrifuge tube, which is the purified hair follicle mesenchymal stem cell exosomes.

[0029] The preparation method of the plant extract comprises the following steps: Platycladus orientalis leaf extract: Dried Platycladus orientalis leaves were ground and soaked in 75% ethanol solution at a solid-liquid ratio of 1g:9mL for 36h. Ultrasonic extraction was performed for 40min, filtered, and the filtrate was concentrated under reduced pressure until there was no alcohol smell to obtain the Platycladus orientalis leaf extract; Polygonum multiflorum extract: After cutting Polygonum multiflorum, reflux extract with 55% ethanol solution at a solid-liquid ratio of 1g:11mL three times, each time for 1.5h. The extracts were combined and concentrated under reduced pressure to obtain the Polygonum multiflorum extract; Ginseng extract: Ginseng was crushed and soaked in 75% ethanol solution at a solid-liquid ratio of 1 g:14 mL for 48 h. The percolation was performed and the percolation liquid was collected and concentrated under reduced pressure to obtain the ginseng extract. Peppermint extract: Peppermint was dried and crushed, and soaked in 45% ethanol solution at a solid-liquid ratio of 1 g:7 mL for 30 h. The mixture was refluxed for 1.5 h, filtered, and the filtrate was concentrated under reduced pressure to obtain the peppermint extract. Purslane extract: Purslane was dried and ground, and refluxed with 75% ethanol solution at a solid-liquid ratio of 1 g:9 mL for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain the purslane extract; Rhodiola rosea extract: After the rhizome of Rhodiola rosea was sliced, it was reflux extracted with 65% ethanol solution at a solid-liquid ratio of 1g:12mL for 3 times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain the Rhodiola rosea extract.

[0030] The preparation method of the hyaluronic acid-collagen peptide graft copolymer comprises the following steps: C1. Dissolve 2 parts of hyaluronic acid in 80 parts of PBS and stir magnetically for 15 minutes to obtain a hyaluronic acid solution. Add 0.4 parts of EDC and 0.18 parts of NHS and activate at 30°C with constant stirring for 1.2 hours. C2. Dissolve 0.8 parts of collagen peptide in 60 parts of deionized water. After complete dissolution, slowly add it dropwise to the above hyaluronic acid solution. React at 35°C under nitrogen protection for 7 hours with magnetic stirring at 150 rpm. C3. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed with deionized water for 50 h, with the deionized water replaced every 6 h. The dialyzed graft copolymer solution is placed in a freeze dryer and freeze-dried at -50 ° C and 0.01 mbar for 24 h to obtain hyaluronic acid-collagen peptide graft copolymer powder, which is sealed and stored for later use; C4. Take 0.8 parts of freeze-dried graft copolymer powder, dissolve it in 80 parts of deionized water to prepare a solution, and use high-pressure homogenization method: 10 cycles of homogenization at a pressure of 800 bar to form a nanoparticle suspension with a particle size of 100-120 nm and a particle size distribution PDI ≤ 0.15.

[0031] The tripeptide is a copper peptide (GHK-Cu), in which the molar ratio of copper ion to tripeptide is 1:1.

[0032] A method for preparing a mesenchymal stem cell hair growth solution comprises the following steps: S1. Take the formulated amount of hyaluronic acid-collagen peptide graft copolymer, add 2 / 3 phosphate buffer solution, and stir on a stirrer until completely dissolved to form an excipient solution; S2. Add Platycladus orientalis leaf extract, Polygonum multiflorum extract, Panax ginseng extract, Mentha piperita extract, Portulaca oleracea extract, and Rhodiola rosea extract to the above-mentioned auxiliary material solution in proportion, and stir at medium speed for 20 minutes; S3. Add vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C, and biotin to the above mixture in proportion, and continue stirring for 15 minutes; S4, sequentially add vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, interleukin-10, tripeptide, and digoxin in proportion, and stir for 12 minutes; S5. Under a sterile environment, slowly add hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes into the above mixture in proportion and gently stir for 8 minutes; S6. Adjust the pH value of the mixture to 7 with phosphate buffer solution, add the remaining phosphate buffer solution to make the mixture reach the specified volume, stir evenly, filter the mixture through a 0.22 μm filter membrane to sterilize, and obtain the final hair follicle mesenchymal stem cell hair growth fluid. Under sterile conditions, divide the hair growth fluid into sterile containers, seal them, and store them at 5°C.

[0033] Example 3 A mesenchymal stem cell hair growth solution, comprising the following components: Hair follicle mesenchymal stem cells 5%; Hair follicle mesenchymal stem cell exosomes 0.05%; Growth factors: vascular endothelial growth factor (VEGF) 0.005%, fibroblast growth factors (FGFs) 0.005%, keratinocyte growth factor (KGF-7) 0.002%, interleukin-10 (IL-10) 0.002%; Vitamins: Vitamin E 0.002%, Vitamin B5 0.001%, Vitamin B2 0.0009%, Vitamin B6 0.0005%, Vitamin C 0.002%, Biotin 0.009%; Plant extracts: Platycladus orientalis leaf extract 3%, Polygonum multiflorum extract 4%, Ginseng extract 2%, Mentha piperita extract 2%, Portulaca oleracea extract 1.5%, Rhodiola rosea extract 0.8%; Other active ingredients: Tripeptide 0.0002%, Digoxin 0.0003%; Excipients: hyaluronic acid-collagen peptide graft copolymer 8%; Balance: phosphate buffer solution.

[0034] Hair follicle mesenchymal stem cells are P5 cells with cell activity ≥95%.

[0035] Fibroblast growth factors (FGFs) include FGF-2 and FGF-7, with a mass ratio of 2:1.

[0036] The method for preparing hair follicle mesenchymal stem cells comprises the following steps: A1, basal medium: DMEM / F12 medium containing L-glutamine and 15 mM HEPES; Supplementary factors: fetal bovine serum (final concentration 5%), 100 U / mL penicillin-streptomycin mixture, 10 ng / mL epidermal growth factor, 5 ng / mL fibroblast growth factor-2, and 50 μg / mL ascorbic acid; Digestion solution: 0.25% trypsin-EDTA; Buffer: PBS, pH 7.4; A2. Take scalp tissue, rinse with PBS three times to remove blood stains, and cut into 1 mm 3 Separate intact hair follicles from small pieces and soak them in PBS containing antibiotics for 10 minutes to sterilize. Use sterile scissors to cut off the upper hair shaft of the hair follicle, retaining the hair bulb containing the dermal papilla and the lower sheath. Transfer the treated hair follicles to 0.25% trypsin-EDTA and digest them at 37°C for 20 minutes. During this period, gently pipette to disperse the cells. Add medium containing 5% FBS to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in basal medium. A3. Adjust the cell density to 5×10 4 Cells were seeded into gelatin-coated 60 mm culture dishes with 0.1% gelatin and incubated at 37°C for 1 hour. The culture medium was not changed within 48 hours, and fresh culture medium was replaced every 3 days thereafter. Cells were passaged when the confluence reached 70-80%. A4. Aspirate and discard the old culture medium, rinse twice with PBS, add 2 mL of trypsin-EDTA, incubate at 37°C for 5 min, add an equal amount of serum-containing culture medium to terminate digestion, gently pipette the bottom of the culture dish to detach the cells, collect the cell suspension, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in fresh culture medium, and inoculate it onto a new gelatin-coated culture dish at a 1:3 ratio; When the confluence of A5 and passage 3 cells reached 50%, they were transferred to a hypoxic incubator with an oxygen concentration of 5% and cultured for 48 h. Wnt-3a was added to the culture medium at a concentration of 20 ng / mL and cultured for 3 consecutive days. A collagen-hyaluronic acid scaffold was used with a concentration of 0.5% and the cells were seeded into the scaffold to form a three-dimensional spheroid culture. A6. Take cells in logarithmic growth phase, digest them and centrifuge them, resuspend the pellet in freezing buffer containing 90% FBS + 10% DMSO, and adjust the density to 1×10 6 / mL, dispense into cryopreservation tubes, store in liquid nitrogen for a long time, and thaw after the cryopreservation tubes are taken out of liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000 rpm for 5 min, discard the cryopreservation solution, resuspend in fresh culture medium and inoculate, and change the medium after 24 h.

[0037] The method for preparing hair follicle mesenchymal stem cell exosomes comprises the following steps: B1. P5 hair follicle mesenchymal stem cells were cultured in a T75 culture flask using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell confluence reached 70-80%, the original medium was discarded and the cells were gently rinsed three times with PBS buffer to remove residual serum. The medium was then replaced with serum-free medium and cultured in a CO2 incubator for 72 hours. B2. Collect the culture supernatant and transfer it to a centrifuge tube. Centrifuge at 300 g for 10 min at 4°C. Then transfer the supernatant to a new centrifuge tube and centrifuge at 2000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane, collect the filtrate, transfer the filtrate to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4000 g for 60 min at 4°C until the volume of the concentrate is 1 / 20 of the original volume. Collect the concentrate. B3. Prepare sucrose gradient solution. Add 60%, 40%, 30%, and 20% sucrose solution to the ultracentrifuge tube in sequence. The volume of each layer of solution is equal. Add gently to avoid disrupting the gradient. Slowly layer the ultrafiltration concentrated sample on the upper layer of the sucrose gradient solution. Ultracentrifuge at 100,000g for 16 hours at 4°C. After centrifugation, use a syringe to collect fractions of different densities from the bottom of the centrifuge tube in sequence, and collect the fraction containing exosomes. B4. Dilute the exosome-containing fraction with PBS buffer and transfer it to a 30 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 g for 30 min at 4°C. Discard the filtrate. Repeat this step three times and collect the concentrated solution in the ultrafiltration centrifuge tube, which is the purified hair follicle mesenchymal stem cell exosomes.

[0038] The preparation method of the plant extract comprises the following steps: Platycladus orientalis leaf extract: Take dried Platycladus orientalis leaves, grind them, and soak them in 80% ethanol solution at a solid-liquid ratio of 1g:10mL for 48 hours. Ultrasonic extraction is performed for 60 minutes. The filtrate is filtered and concentrated under reduced pressure until there is no alcohol taste to obtain the Platycladus orientalis leaf extract; Polygonum multiflorum extract: After cutting Polygonum multiflorum, reflux extract with 60% ethanol solution at a solid-liquid ratio of 1g:12mL three times, each time for 2 hours. The extracts were combined and concentrated under reduced pressure to obtain the Polygonum multiflorum extract; Ginseng extract: Ginseng was crushed and soaked in 80% ethanol solution at a solid-liquid ratio of 1 g:15 mL for 48 h. The percolation was performed and the percolation liquid was collected and concentrated under reduced pressure to obtain the ginseng extract. Peppermint extract: Peppermint was dried and crushed, and soaked in 50% ethanol solution at a solid-liquid ratio of 1 g:8 mL for 36 h. The mixture was refluxed for 2 h, filtered, and the filtrate was concentrated under reduced pressure to obtain the peppermint extract. Purslane extract: Purslane was dried and ground, and refluxed with 80% ethanol solution at a solid-liquid ratio of 1 g:10 mL for three times, each time for 2.5 hours. The extracts were combined and concentrated under reduced pressure to obtain the purslane extract; Rhodiola rosea extract: After the rhizome of Rhodiola rosea was sliced, it was reflux extracted with 70% ethanol solution at a solid-liquid ratio of 1g:15mL for 3 times, each time for 3 hours. The extracts were combined and concentrated under reduced pressure to obtain the Rhodiola rosea extract.

[0039] The preparation method of the hyaluronic acid-collagen peptide graft copolymer comprises the following steps: C1. Dissolve 3 parts of hyaluronic acid in 100 parts of PBS and stir magnetically for 30 minutes to obtain a hyaluronic acid solution. Add 0.5 parts of EDC and 0.2 parts of NHS and activate at 30°C with constant stirring for 1.5 hours. C2. Dissolve 1 part of collagen peptide in 80 parts of deionized water. After complete dissolution, slowly add it dropwise to the above hyaluronic acid solution. React at 35°C under nitrogen protection for 8 hours with magnetic stirring at 200 rpm. C3. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed with deionized water for 72 h, with the deionized water replaced every 6 h. The dialyzed graft copolymer solution is placed in a freeze dryer and freeze-dried at -50 ° C and 0.01 mbar for 24 h to obtain hyaluronic acid-collagen peptide graft copolymer powder, which is sealed and stored for later use; C4. Take 1 part of the freeze-dried graft copolymer powder and dissolve it in 100 parts of deionized water to prepare a solution. Then use high-pressure homogenization method: 10 cycles of homogenization at a pressure of 800 bar to form a nanoparticle suspension with a particle size of 100-120 nm and a particle size distribution PDI ≤ 0.15.

[0040] The tripeptide is a copper peptide (GHK-Cu), in which the molar ratio of copper ion to tripeptide is 1:1.

[0041] A method for preparing a mesenchymal stem cell hair growth solution comprises the following steps: S1. Take the formulated amount of hyaluronic acid-collagen peptide graft copolymer, add 2 / 3 phosphate buffer solution, and stir on a stirrer until completely dissolved to form an excipient solution; S2. Add Platycladus orientalis leaf extract, Polygonum multiflorum extract, Panax ginseng extract, Mentha piperita extract, Portulaca oleracea extract, and Rhodiola rosea extract to the above-mentioned auxiliary material solution in proportion, and stir at medium speed for 30 minutes; S3. Add vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C, and biotin to the above mixture in proportion, and continue stirring for 20 minutes; S4, sequentially add vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, interleukin-10, tripeptide, and digoxin in proportion, and stir for 15 minutes; S5. Under a sterile environment, slowly add hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes into the above mixture in proportion and gently stir for 10 minutes; S6. Adjust the pH value of the mixture to 7.2 with phosphate buffer solution, add the remaining phosphate buffer solution to make the mixture reach the specified volume, stir evenly, filter the mixture through a 0.22 μm filter membrane to sterilize, and obtain the final hair follicle mesenchymal stem cell hair growth fluid. Under sterile conditions, divide the hair growth fluid into sterile containers, seal them, and store them at 8°C.

[0042] Comparative Example 1: Hair follicle mesenchymal stem cells were not included, and the remaining ingredients and process were the same as those in Example 1.

[0043] Comparative Example 2: The hair follicle mesenchymal stem cell exosomes were not included, and the remaining ingredients and process were the same as those in Example 1.

[0044] Comparative Example 3: No growth factor was included, and the remaining ingredients and process were the same as in Example 1.

[0045] Comparative Example 4: No vitamins or plant extracts are included, and the remaining ingredients and process are the same as in Example 1.

[0046] Comparative Example 5: The hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes were not included, and the remaining ingredients and process were the same as those in Example 1.

[0047] Hair growth test: 1. Animal Experimentation 1. Experimental Model A hair loss model was established by depilating the back of 6-8 week-old C57BL / 6 male mice (10 mice per group). The mice were treated with the sample once a day for 4 consecutive weeks starting from the first day after depilation.

[0048] 2. Test indicators and methods (1) Hair growth area Detection method: The hair removal area on the back was photographed weekly with a digital camera, and the percentage of hair coverage area to the total hair removal area was calculated using Image-Pro Plus software. (2) Proportion of hair follicle growth phase (3) Hair follicle density Detection method: In HE-stained sections, count the number of 2 The number of hair follicles in the skin (5 fields of view were counted in each group and the average value was taken). (4) Vascular density Detection method: CD31 immunohistochemical staining was used to mark vascular endothelial cells in skin tissue and count the number of cells per mm 2 The number of blood vessels in the skin (5 fields of view were counted in each group and the average value was taken). (5) Levels of inflammatory factors Detection method: Skin tissue homogenate was obtained and the IL-6 (pro-inflammatory factor) content was detected using an ELISA kit (unit: pg / mg protein).

[0049] Table 1 Test results ; As can be seen from the above table, the various indicators of Examples 1 to 3 are significantly better than those of Comparative Examples 1 to 5, indicating that stem cells and their exosomes, growth factors, vitamins and plant extracts can promote hair follicle activation and angiogenesis, reduce inflammatory responses, and have a significant hair growth effect.

[0050] 2. Clinical Trials 1. Test subjects A total of 300 patients with androgenic alopecia aged 18-60 years (conforming to Norwood-Hamilton grade II-V) were selected and randomly divided into 6 groups (50 cases in each group). There were no significant differences in baseline data (age, degree of alopecia, and course of disease) among the groups (P>0.05), ensuring comparability.

[0051] 2. Usage All subjects each morning and evening, 1 times, the hair growth liquid is evenly sprayed on the hair loss area (0.5mL each time), massage 30s to promote absorption, continuous use for 6 months, during which other hair growth products are prohibited.

[0052] 3. Detection method (1) Hair density (root / cm 2 ) Detection method: Using hair detector (precision 0.01mm) in fixed detection area (top of the head before hairline 3cm x 3cm area) to take pictures, computer automatically count the number of hair per cm 2 Hair quantity, calculate the number of hair density increased after 6 months compared with baseline (root / cm 2 ).

[0053] (2) Change of hair diameter (μm) Detection method: The same hair detector measures the diameter of 100 terminal hairs in the fixed area, takes the average value, and calculates the percentage increase of hair diameter after 6 months compared with baseline. (3) Patient satisfaction score Evaluation criteria: Using visual analogue scale (VAS), 0 points (no improvement) to 10 points (significant improvement), scored by patients themselves. (4) Safety index Record the incidence of skin irritation (erythema, itching, desquamation) during use, follow up once a week.

[0054] Table 2 Test results ; From the above table, the hair diameter growth and hair density growth of examples 1-3 are higher than those of examples 1-3, indicating that the hair growth liquid of examples 1-3 has more obvious hair growth effect, and the patients are more satisfied with the effect of the hair growth liquid produced by examples, and the skin irritation rate is also lower.

[0055] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A mesenchymal stem cell hair growth fluid, characterized in that: It is composed of the following ingredients: Hair follicle mesenchymal stem cells 2~5%; Hair follicle mesenchymal stem cell exosomes 0.02~0.05%; Growth factors: vascular endothelial growth factor (VEGF) 0.002~0.005%, fibroblast growth factors (FGFs) 0.003~0.005%, keratinocyte growth factor (KGF-7) 0.001~0.002%, interleukin-10 (IL-10) 0.001~0.002%; Vitamins: Vitamin E 0.001~0.002%, Vitamin B5 0.0008~0.001%, Vitamin B2 0.0006~0.0009%, Vitamin B6 0.0002~0.0005%, Vitamin C 0.001~0.002%, Biotin 0.007~0.009%; Plant extracts: Platycladus orientalis leaf extract 1-3%, Polygonum multiflorum extract 2-4%, Ginseng extract 0.5-2%, Peppermint extract 0.5-2%, Portulaca oleracea extract 0.8-1.5%, Rhodiola rosea extract 0.6-0.8%; Other active ingredients: Tripeptide 0.00015~0.0002%, Digoxin 0.0001~0.0003%; Excipients: hyaluronic acid-collagen peptide graft copolymer 5~8%; Balance: phosphate buffer solution.

2. The mesenchymal stem cell hair growth liquid according to claim 1, characterized in that: The method for preparing hair follicle mesenchymal stem cells comprises the following steps: A1, basal medium: DMEM / F12 medium containing L-glutamine and 15 mM HEPES; Supplementary factors: fetal bovine serum (final concentration 5%), 100 U / mL penicillin-streptomycin mixture, 10 ng / mL epidermal growth factor, 5 ng / mL fibroblast growth factor-2, and 50 μg / mL ascorbic acid; Digestion solution: 0.25% trypsin-EDTA; Buffer: PBS, pH 7.2–7.4; A2. Take scalp tissue, rinse with PBS three times to remove blood stains, and cut into 1 mm 3 Separate intact hair follicles from small pieces and soak them in PBS containing antibiotics for 10 minutes to sterilize. Use sterile scissors to cut off the upper hair shaft of the hair follicle, retaining the hair bulb containing the dermal papilla and the lower sheath. Transfer the treated hair follicles to 0.25% trypsin-EDTA and digest them at 37°C for 20 minutes. During this period, gently pipette to disperse the cells. Add medium containing 5% FBS to terminate the digestion. Centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and resuspend the pellet in basal medium. A3. Adjust the cell density to 5×10 4 Cells were seeded into gelatin-coated 60 mm culture dishes with 0.1% gelatin and incubated at 37°C for 1 hour. The culture medium was not changed within 48 hours, and fresh culture medium was replaced every 3 days thereafter. Cells were passaged when the confluence reached 70-80%. A4. Aspirate and discard the old culture medium, rinse twice with PBS, add 2 mL of trypsin-EDTA, and incubate at 37°C for 3–5 min. Add an equal amount of serum-containing culture medium to terminate digestion. Gently pipette the bottom of the culture dish to dislodge the cells. Collect the cell suspension and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend the pellet in fresh culture medium, and inoculate it onto a new gelatin-coated culture dish at a 1:3 ratio. When the confluence of A5 and passage 3 cells reached 50%, they were transferred to a hypoxic incubator with an oxygen concentration of 5% and cultured for 48 h. Wnt-3a was added to the culture medium at a concentration of 20 ng / mL and cultured for 3 consecutive days. A collagen-hyaluronic acid scaffold was used with a concentration of 0.5% and the cells were seeded into the scaffold to form a three-dimensional spheroid culture. A6. Take cells in logarithmic growth phase, digest them and centrifuge them, resuspend the pellet in freezing buffer containing 90% FBS + 10% DMSO, and adjust the density to 1×10 6 / mL, dispense into cryopreservation tubes, store in liquid nitrogen for a long time, and thaw after the cryopreservation tubes are taken out of liquid nitrogen, thaw quickly in a 37℃ water bath, centrifuge at 1000 rpm for 5 min, discard the cryopreservation solution, resuspend in fresh culture medium and inoculate, and change the medium after 24 h.

3. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: Hair follicle mesenchymal stem cells are P3~P5 cells, and the cell activity is ≥95%.

4. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: The method for preparing hair follicle mesenchymal stem cell exosomes comprises the following steps: B1. Take P3-P5 hair follicle mesenchymal stem cells and culture them in a T75 culture flask using DMEM / F12 medium supplemented with 10% fetal bovine serum. When the cell confluence reaches 70-80%, discard the original medium and gently rinse the cells 2-3 times with PBS buffer to remove residual serum. Then replace the medium with serum-free medium and continue to culture in a CO2 incubator for 48-72 hours. B2. Collect the culture supernatant and transfer it to a centrifuge tube. Centrifuge at 300 g for 10 min at 4°C. Then transfer the supernatant to a new centrifuge tube and centrifuge at 2000 g for 10 min. Filter the supernatant through a 0.22 μm filter membrane, collect the filtrate, transfer the filtrate to a 30 kDa ultrafiltration centrifuge tube, and centrifuge at 4000 g for 30-60 min at 4°C until the volume of the concentrate is 1 / 10-1 / 20 of the original volume. Collect the concentrate. B3. Prepare sucrose gradient solution. Add 60%, 40%, 30%, and 20% sucrose solution to the ultracentrifuge tube in sequence. The volume of each layer of solution is equal. Add gently to avoid disrupting the gradient. Slowly layer the ultrafiltration concentrated sample on the upper layer of the sucrose gradient solution. Ultracentrifuge at 100,000g for 16 hours at 4°C. After centrifugation, use a syringe to collect fractions of different densities from the bottom of the centrifuge tube in sequence, and collect the fraction containing exosomes. B4. Dilute the exosome-containing fraction with PBS buffer and transfer it to a 30 kDa ultrafiltration centrifuge tube. Centrifuge at 4000 g for 30 min at 4°C. Discard the filtrate. Repeat this step 2-3 times and collect the concentrated solution in the ultrafiltration centrifuge tube, which is the purified hair follicle mesenchymal stem cell exosomes.

5. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: Fibroblast growth factors (FGFs) include FGF-2 and FGF-7, with a mass ratio of 2:

1.

6. The mesenchymal stem cell hair growth liquid according to claim 1, characterized in that: The preparation method of the plant extract comprises the following steps: Platycladus orientalis leaf extract: Take dried Platycladus orientalis leaves, grind them, and soak them in 70-80% ethanol solution at a solid-liquid ratio of 1g:8-10mL for 24-48 hours. Ultrasonic extraction is performed for 30-60 minutes. The filtrate is filtered and concentrated under reduced pressure until there is no alcohol taste to obtain the Platycladus orientalis leaf extract. Polygonum multiflorum extract: After cutting Polygonum multiflorum, reflux extract it with 50-60% ethanol solution at a material-liquid ratio of 1 g: 10-12 mL for 2-3 times, each time for 1-2 hours. Combine the extracts and concentrate under reduced pressure to obtain the Polygonum multiflorum extract; Ginseng extract: After crushing ginseng, soak it in 70-80% ethanol solution at a material-liquid ratio of 1g:12-15mL for 36-48h, extract by percolation, collect the percolation liquid, and concentrate under reduced pressure to obtain the ginseng extract; Peppermint extract: Peppermint is dried and crushed, and soaked in a 40-50% ethanol solution at a solid-liquid ratio of 1 g:6-8 mL for 24-36 hours. Reflux extraction is performed for 1-2 hours, filtered, and the filtrate is concentrated under reduced pressure to obtain the peppermint extract. Purslane extract: Purslane is dried and crushed, and reflux-extracted with 70-80% ethanol solution at a solid-liquid ratio of 1 g:8-10 mL for 2-3 times, each time for 1.5-2.5 hours. The extracts are combined and concentrated under reduced pressure to obtain the purslane extract; Rhodiola rosea extract: After the rhizome of Rhodiola rosea is sliced, it is refluxed and extracted 2 to 3 times with 60-70% ethanol solution at a solid-liquid ratio of 1 g:10-15 mL, each time for 2 to 3 hours. The extracts are combined and concentrated under reduced pressure to obtain the Rhodiola rosea extract.

7. The mesenchymal stem cell hair growth fluid according to claim 1, characterized in that: The preparation method of the hyaluronic acid-collagen peptide graft copolymer comprises the following steps: C1. Dissolve 1-3 parts of hyaluronic acid in 50-100 parts of PBS and stir magnetically for 10-30 minutes to obtain a hyaluronic acid solution. Add 0.3-0.5 parts of EDC and 0.15-0.2 parts of NHS and activate at 30°C with constant stirring for 1-1.5 hours. C2. Dissolve 0.5-1 part collagen peptide in 50-80 parts deionized water. After complete dissolution, slowly add it dropwise to the above hyaluronic acid solution. React at 35°C under nitrogen protection for 6-8 hours. Maintain magnetic stirring at 100-200 rpm during this period. C3. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 10 kDa and dialyzed with deionized water for 48-72 hours, with the deionized water replaced every 6 hours. The dialyzed graft copolymer solution is placed in a freeze dryer and freeze-dried at -50°C and 0.01 mbar for 24 hours to obtain hyaluronic acid-collagen peptide graft copolymer powder, which is then sealed and stored for later use. C4. Take 0.5-1 parts of freeze-dried graft copolymer powder and dissolve it in 50-100 parts of deionized water to prepare a solution. Then use high-pressure homogenization method: 10 cycles of homogenization at 800 bar pressure to form a nanoparticle suspension with a particle size of 100-120 nm and a particle size distribution (PDI) ≤ 0.

15.

8. The mesenchymal stem cell hair growth liquid according to claim 1, characterized in that: The tripeptide is a copper peptide (GHK-Cu), in which the molar ratio of copper ion to tripeptide is 1:

1.

9. The method for preparing a mesenchymal stem cell hair growth solution according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. Take the formulated amount of hyaluronic acid-collagen peptide graft copolymer, add 2 / 3 phosphate buffer solution, and stir on a stirrer until completely dissolved to form an excipient solution; S2. Add Platycladus orientalis leaf extract, Polygonum multiflorum extract, Panax ginseng extract, Mentha piperita extract, Portulaca oleracea extract, and Rhodiola rosea extract to the above-mentioned auxiliary material solution in proportion, and stir at medium speed for 10 to 30 minutes; S3. Add vitamin E, vitamin B5, vitamin B2, vitamin B6, vitamin C, and biotin to the above mixture in proportion, and continue stirring for 10-20 minutes; S4. Add vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, interleukin-10, tripeptide, and digoxin in proportion and stir for 10-15 minutes; S5. Under a sterile environment, slowly add hair follicle mesenchymal stem cells and hair follicle mesenchymal stem cell exosomes into the above mixture in proportion and gently stir for 5-10 minutes; S6. Adjust the pH value of the mixture to 6.8-7.2 with phosphate buffer solution, add the remaining phosphate buffer solution to make the mixture reach the specified volume, stir evenly, filter the mixture through a 0.22 μm filter membrane to sterilize, and obtain the final hair follicle mesenchymal stem cell hair growth fluid. Under sterile conditions, divide the hair growth fluid into sterile containers, seal them, and store them at 2-8°C.