New uses of the serpine1 gene, the protein it codes for or active derivatives thereof

Abstract

This invention discloses novel applications of the SERPINE1 gene, its encoded protein, or its active derivatives, belonging to the field of biology. It discloses the application of the SERPINE1 gene, its encoded protein, or its active derivatives as active ingredients in the preparation of products that promote hair growth and / or hair follicle regeneration, treat hair loss, and in hair follicle organoid models. Compared to existing technologies, this method has novel targets and a clear mechanism, and its effectiveness has been verified in both in vivo and in vitro models, providing a reliable foundation for developing research protocols and application strategies to promote hair growth.

Description

Technical Field

[0001] This invention belongs to the field of biology, and specifically relates to a new application of the SERPINE1 gene, the protein it encodes, or its active derivatives. Background Technology

[0002] Mammalian hair follicles undergo a continuous cyclical process (anagen, catagen, telogen) after birth, a process precisely regulated by complex interactions between epithelial and mesenchymal cells (such as dermal papillary cells). The synchronicity (e.g., in mice) or asynchronicity (e.g., in humans) of the hair follicle cycle makes it a classic model for studying organ regeneration and stem cell behavior. Cycle transitions involve the coordination of cell proliferation, apoptosis, differentiation, and signal transduction, and are regulated by a multi-level network of signaling pathways (such as Wnt / β-catenin, BMP, FGF, IGF, TGF-β, etc.), transcription factors, and epigenetic modifiers. For example, sustained activation of Wnt / β-catenin signaling is essential for maintaining the anagen phase of the hair follicle, while FGF5 has been shown to participate in the termination of the anagen phase. Although many signaling molecules have been shown to be involved in this process, the precise upstream triggers and complete regulatory network of hair follicle cycle initiation and transition remain incompletely elucidated. In particular, key regulatory molecules that can effectively drive telogen follicles back into the active anagen phase remain a focus of research and technological development.

[0003] The SERPINE1 (Plasminogen Activator Inhibitor-1) gene encodes a serine protease inhibitor and has traditionally been extensively studied in areas such as fibrinolytic system regulation, thrombosis, tissue fibrosis, and cancer progression. It affects extracellular matrix (ECM) degradation, cell adhesion, migration, and various intracellular signaling pathways (such as the JAK / STAT pathway) by inhibiting urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In skin biology, SERPINE1 has been reported to participate in keratinocyte migration and skin wound healing. However, to date, whether the SERPINE1 gene directly participates in and regulates the postnatal hair follicle cycle, its specific functional role in the hair follicle cycle transition, and whether it can be used as a key factor in hair follicle regeneration models remain unclear and lacks well-established methods. Summary of the Invention

[0004] The purpose of this invention is to provide the SERPINE1 gene, its encoded protein, or its active derivatives as active ingredients in the preparation of products that promote hair growth and / or hair follicle regeneration, treat hair loss, and in hair follicle organoid models. This aims to address the limitations of existing hair follicle regeneration models in terms of limited growth induction efficiency and insufficient regulatory mechanisms.

[0005] The objective of this invention is achieved through the following technical solutions: The use of the SERPINE1 gene, its encoded protein, or its active derivatives as active ingredients in the preparation of products that promote hair growth and / or hair follicle regeneration and / or treat hair loss.

[0006] Preferably, the product includes any one or a combination of cosmetics, skin care products, pharmaceuticals, or medical devices.

[0007] Preferably, the medical device is a microneedle, a microneedle array, or a biodegradable implantable material.

[0008] Preferably, the SERPINE1 gene is delivered via adenovirus, lentivirus, adeno-associated virus, or retroviral vector.

[0009] Preferably, the drug or medical device is used to promote the transition of hair follicles from the resting phase to the growth phase, and / or accelerate the hair follicle cycle.

[0010] Preferably, the drug or medical device is used to prevent or treat androgenetic alopecia, alopecia areata, or telogen effluvium.

[0011] Preferably, cosmetics and skin care products are used to promote hair growth or improve sparse hair.

[0012] Preferably, the protein encoded by SERPINE1 or its active derivatives promote hair growth or hair follicle regeneration by regulating the PI3K / AKT signaling pathway.

[0013] The present invention also provides the use of cells containing the SERPINE1 gene or its encoded protein in the preparation of a hair growth promoting agent, wherein the cells stably express or overexpress the SERPINE1 gene or its active derivative.

[0014] Compared with the prior art, the present invention has the following technical effects: In the technical solution provided by this invention, by specifically activating the SERPINE1 gene function in a resting phase mouse model and a hair follicle organoid model, the hair growth process is significantly accelerated, and the hair follicle transition from the resting phase to the anagen phase is effectively driven. By setting up an inhibitor control group, it was clarified that this promoting effect depends on the activation of the PI3K / AKT signaling pathway. The growth-promoting effect of the SERPINE1 gene can be completely blocked using the inhibitor LY294002. This invention establishes a method for promoting hair follicle regeneration with the SERPINE1 gene as a key regulatory factor, providing a new and effective model and tool for studying hair follicle cycle regulation. Compared with existing technologies, this method has a novel target and a clear mechanism, and its effectiveness has been verified in both in vivo and in vitro models, providing a reliable foundation for developing research programs and application strategies to promote hair growth.

[0015] The discovery of the role of the SERPINE1 gene in the regulation of the hair follicle cycle not only provides a new molecular perspective for understanding the biology of hair follicle regeneration, but also offers a novel target for developing new tools and strategies to promote hair growth and regulate the hair follicle cycle. This can be used to develop drugs or biologics that promote hair regeneration. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 A diagram showing the effect of Western blotting on SERPINE1 gene overexpression; Figure 2 To observe the growth of hair on the back of mice at different time points after experimental treatment; Figure 3 Image showing HE staining results of mouse hair follicles after experimental treatment; Figure 4 This is an image showing the results of immunofluorescence staining of mouse hair follicle skin tissue after experimental treatment; Figure 5 To observe the culture status of hair follicle organoids at different time points after experimental treatment; Figure 6 This is a diagram showing the results of immunofluorescence staining of hair follicle organoids after experimental treatment.

[0018] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0020] Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or a solution that simultaneously satisfies A and B. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or impossible to implement, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0021] To investigate the function of the SERPINE1 gene, this invention uses an adenovirus overexpression system to conduct gain-of-function studies.

[0022] Example 1 Construction of a SERPINE1 overexpression adenovirus vector Based on the goat SERPINE1 gene sequence published on NCBI, after sequence alignment, the complete CDS region sequence of the SERPINE1 gene (1233 bp) was specifically amplified by PCR using XhoI and KpnI restriction endonucleases as digestion sites and cDNA from Shaanbei white cashmere goat skin tissue as a template. The correctly amplified PCR product was then purified and recovered using a gel extraction kit (Omega, USA). The gel-extracted product and the pAdTrack-CMV empty vector were digested with XhoI and KpnI, respectively, at 37°C for 3-4 hours, followed by purification and recovery. The gel-extracted target fragment and the empty vector were ligated and incubated overnight at 16°C in a PCR instrument. Subsequently, 5 μL of the ligation product was added to 100 μL of pre-allocated *E. coli* DH5α competent cells. After incubating on ice for 30 min, the cells were rapidly transferred to a 42°C water bath for 65 s heat shock, followed by a rapid transfer to ice for 10 min. The ligation product was then evenly spread on preheated (37°C) culture dishes and incubated at 37°C for 10–12 h until single colonies formed on the solid medium. Single colonies of appropriate size were picked using a sterilized pipette tip and placed in a test tube containing 5 mL of kanamycin-resistant liquid medium. The tube was tilted and incubated at 250 rpm and 37°C for approximately 12 h. Plasmid extraction was performed after the liquid in the test tube became turbid and the pipette tip was no longer visible. The pAd-track-CMV-SERPINE1 recombinant vector was successfully constructed. Based on this, the vector was linearized using Pme I enzyme for 3 hours, and then successfully linearized vector was transformed into BJ5183 competent cells to obtain recombinant adenovirus vector pAdEasy-SERPINE1. A blank vector was used as the control group, pAd-NC. Subsequently, the Pac I-linearized recombinant adenovirus vector was transfected into HEK293A cells for virus packaging. A typical "comet tail" phenomenon was observed 5 days after transfection, and almost all cells glowed green around day 7. When all cells glowed green and some cells detached, the recombinant adenovirus was collected, named pAd-SERPINE1, and stored at -80℃ for later use.

[0023] To verify the overexpression effect, HFSCs were infected with pAd-SERPINE1 and pAd-NC. RT-qPCR and WB results showed that, compared with the control group, the expression level of SERPINE1 in the experimental group was significantly increased ( Figure 1 This indicates that the adenovirus overexpression system was successfully constructed and the overexpression effect was good.

[0024] Example 2 Effects of SERPINE1-PI3K / AKT axis on hair growth in mice 1. Preparation of experimental animals and establishment of the model: Nine healthy male C57BL / 6 mice, 21 days old (hair follicles entering the resting phase), were selected. This strain of mice has a highly synchronized hair cycle, making observation easy. The mice were first anesthetized by intraperitoneal injection of sodium pentobarbital at 0.2 ml / 10 g. After anesthesia, the backs of all mice were shaved and cleaned with warm water.

[0025] 2. Experimental grouping and preparation of treatment reagents (1) Experimental grouping: Mice were randomly divided into the following 3 groups, with 3 mice in each group: ① Negative control group (pAd-NC group): injected with empty control adenovirus; ② SERPINE1 overexpression group (pAd-SERPINE1 group): Injection of recombinant adenovirus carrying the SERPINE1 gene prepared in Example 1; ③ Pathway inhibition group (pAd-SERPINE1 + LY294002 group): combined injection of pAd-SERPINE1 adenovirus and PI3K inhibitor LY294002.

[0026] (2) Reagent preparation: ① Adenovirus solution: The pAd-NC and pAd-SERPINE1 stock solutions were diluted to the working titer with sterile physiological saline. In this study, based on the adenovirus titer determination, 50 μl of adenovirus was dissolved in 200 μl of physiological saline, for a total of 250 μl. After aliquoting, the solutions were stored at -80℃ and thawed on ice before use.

[0027] ② Inhibitor solution: Dissolve LY294002 powder in dimethyl sulfoxide (DMSO) to prepare a stock solution. When using, further dilute with PBS containing 1% DMSO to the working concentration (10 μM).

[0028] 3. Experimental Treatment ① Injection time: The first subcutaneous injection will be performed on day 0 after hair removal (referred to as Day 0).

[0029] ② Injection method: After fixing the mouse, use a 1mL syringe to inject subcutaneously at multiple points in the hairless area on the back, with a total injection volume of 250μL at each point, to ensure that the virus or inhibitor solution is evenly distributed in the target area.

[0030] 4. Housing conditions: After injection, mice were housed in standard specific pathogen-free (SPF) animal rooms with constant temperature (22±2℃), humidity (50±10%) and 12-hour light / dark cycle, and free access to food and water.

[0031] 5. Observation and Phenotypic Records ① Macroscopic observation: Starting from the 5th day after the first injection, observe and photograph the hair regeneration on the back of the mice daily or every other day.

[0032] ② Quantitative assessment: Use image analysis software (such as ImageJ) to analyze the photos, calculate the percentage of hair coverage area to total hair removal area at each time point, plot hair regeneration dynamics curves, and compare the differences in growth rate among groups.

[0033] 6. Tissue Sample Collection and Processing ① Timing of sample collection: Sample collection can be carried out at the time when the difference in hair regeneration is most significant (e.g., 10-15 days after treatment).

[0034] ② Sampling: Mice were euthanized by cervical dislocation, and the skin tissue in the treated area on the back was completely dissected using surgical scissors and forceps.

[0035] ③ Sample allocation: Skin tissue was immediately placed in 4% paraformaldehyde phosphate buffer and fixed at 4°C for 24 hours for subsequent paraffin embedding and histological analysis.

[0036] 7. Histological analysis (HE staining) ①Preparation of paraffin sections: a. Washing: Wrap the fixed skin tissue with gauze, put it in a beaker, and place the beaker under a tap. Let the slow flow of water through the rubber tube attached to the tap flow into the beaker for rinsing for 2-3 hours. b. Dehydration: Skin tissue was immersed in 70% anhydrous ethanol, 80% anhydrous ethanol, 90% anhydrous ethanol, 100% anhydrous ethanol I and 100% anhydrous ethanol II for 2 hours respectively, and then immersed in a 1:1 solution of xylene and 100% anhydrous ethanol for more than 15 minutes; c. Transparent: Exposed to xylene twice, 10 minutes each time; d. Wax immersion: Immerse the skin tissue in melted wax for at least 3 hours; e. Paraffin embedding: Skin tissue is immersed in paraffin in an embedding cassette. Then, it is continuously sectioned perpendicular to the skin surface, with a thickness of 5 μm.

[0037] ② Hematoxylin-eosin (HE) staining: Dewaxing: Immerse in xylene I for 10 minutes, then immerse in xylene II for 10 minutes; b. Rehydration: Soak in 100% anhydrous ethanol I, 100% anhydrous ethanol II, 95% anhydrous ethanol, 90% anhydrous ethanol, 80% anhydrous ethanol, 70% anhydrous ethanol and 50% anhydrous ethanol for 5 min respectively, and then soak in PBS for 2 min. c. Staining: Stain with hematoxylin solution for 5-15 minutes. Rinse with running water to regain blue color. Then soak in 1% hydrochloric acid alcohol for 1-2 seconds, rinse with running water for 5 minutes. Stain with eosin solution for 1-3 minutes; d. Dehydration: After soaking in PBS for 2 min, soak in 50% anhydrous ethanol, 70% anhydrous ethanol, 80% anhydrous ethanol, 90% anhydrous ethanol, 100% anhydrous ethanol I and 100% anhydrous ethanol II for 5 min respectively; e. Transparent: Soak in xylene I for 10 minutes, then soak in xylene II for 10 minutes; f. Mounting: Mount the slide with neutral resin. The amount added depends on the area of ​​the tissue on the slide; generally, 5-10 μL is sufficient. Observe under a microscope.

[0038] ③ Immunofluorescence staining of tissues a. Dewaxing: First, place the sections in a 60-degree oven for 2 hours, then expose them to xylene twice, 15 minutes each time, to remove the paraffin wax from the sections. b. Rehydration: Soak in 100% anhydrous ethanol, 95% anhydrous ethanol, 90% anhydrous ethanol, 80% anhydrous ethanol, 70% anhydrous ethanol and 50% anhydrous ethanol for 5 min respectively, and then soak in PBS for 10 min. c. Antigen retrieval: After rehydration, place the rehydrated slides in a citric acid antigen retrieval solution heated to 96 degrees Celsius and boil for 10 minutes. After the retrieval solution cools to room temperature, remove the slides and wash them with TBS and TBST for 5 minutes each. d. Sealing: Add approximately 50 μl of BDT, then seal the section with a sealing film. Incubate at room temperature for 30 minutes. Label the area to prevent accidental tampering. e. Primary antibody incubation: Remove the sealing film and pour any excess blocking solution onto absorbent paper. Add 20 μL of primary antibody to the section location. Seal the primary antibody with a clean coverslip, ensuring there are no air bubbles, especially at the tissue section location. Place the slide in a humidified chamber, seal it with a sealing bag, and incubate overnight at 4°C. After primary antibody incubation, remove the slide and immerse it in TBST. Wash three times in total, for 10 min, 20 min, and 20 min respectively. The coverslip must be removed by immersion in TBST; do not forcibly remove it directly with tweezers.

[0039] The following steps are all completed in a darkroom: f. Incubation with secondary antibody: Pour TBST from the slide onto absorbent paper, add 20 μL of secondary antibody diluted with BDT. Seal the secondary antibody with sealing film, place in a humidified chamber, seal with a sealing bag, and incubate at 37°C for 30 min. After incubation, remove the slide, peel off the sealing film, and wash three times with TBST for 10 min, 20 min, and 20 min respectively. g. Counterstaining cell nuclei: Pour excess TBST from the slide onto absorbent paper and add 10 μL of Hoechst 33342. Cover the slide with a sealing film and incubate at room temperature for 4 minutes. Staining of the slide should be significantly faster than that of the cells. Remove the sealing film, place the slide in PBS, and rinse 4 times, each time for no more than 1 second. h. Mounting: Pour excess PBS onto absorbent paper. Add Vectashield (anti-quencher) to the slide for mounting. The amount added depends on the area of ​​the tissue on the slide; generally, 5-10 μL is sufficient. Observe under a fluorescence microscope.

[0040] Based on in vivo experimental verification, this invention provides a clear and reproducible new use of the SERPINE1 gene in promoting hair growth and regulating the hair follicle cycle, and elucidates its core mechanism of action.

[0041] By establishing a C57BL / 6 mouse model and conducting rigorous grouped control experiments (pAd-NC group, pAd-SERPINE1 group, pAd-SERPINE1 + LY294002 group), this invention confirms that overexpression of the SERPINE1 gene can significantly accelerate the regeneration rate of hair on the back of mice, with significant macroscopic phenotypic differences. Key mechanism studies show that the combination with the specific inhibitor LY294002 can completely reverse the promoting effect of the SERPINE1 gene, indicating that this growth-promoting effect depends on the activation of the PI3K / AKT signaling pathway. Figure 2 Further microscopic histological analysis (HE staining) and immunofluorescence analysis revealed that overexpression of the SERPINE1 gene effectively drives the hair follicle to transition from the resting phase to the anagen phase (during the anagen phase, the hair follicle root (hair bulb) is swollen and tightly wraps around the dermal papilla. This is the core area where hair matrix cells rapidly divide and push the hair shaft upwards. The hair shaft grows upwards, with its root deeply embedded within the hair bulb). Inhibition of PI3K activity, on the other hand, causes the hair follicle to remain in the resting phase (during the resting phase, the hair bulb disappears, replaced by a compact cluster of cells called the secondary hair bud. The dermal papilla eventually remains in the dermis, directly below or beside the secondary hair bud, its morphology changing from the loose, pear-shaped structure of the anagen phase to a dense, small spherical shape. The overall length of the hair follicle is significantly shortened, and the secondary hair bud and dermal papilla retract and remain in the dermis. At this point, the structural connection between the hair follicle and the subcutaneous fat layer is lost). Figure 3 and Figure 4 ).

[0042] Following SERPINE1 gene overexpression, hair follicles enter the anagen phase on day 10 (as opposed to...). Figure 2 The result has been consistent. Figure 2The gray-black skin on the backs of the mice indicates that the hair follicles have entered the growth phase. However, the NC group and the group that simultaneously added SERPINE1 gene overexpression adenovirus and LY294002, an inhibitor of the PI3K / AKT signaling pathway, were still in the resting phase on day 10. This proves that the SERPINE1 gene can promote the hair follicles to enter the growth phase earlier, and that the SERPINE1 gene depends on the PI3K / AKT signaling pathway to function.

[0043] In summary, this invention not only discovers and verifies that the SERPINE1 gene is a novel and effective hair growth promoter, but more importantly, it clearly reveals for the first time in an in vivo model the core role of the "SERPINE1-PI3K / AKT" signaling axis in regulating hair follicle cycle transition. This discovery provides a solid experimental basis and a clear mechanism of action for developing novel hair growth regulation strategies (including drugs, biologics, and cosmetic active ingredients) targeting the SERPINE1 gene.

[0044] Example 3 The effect of the SERPINE1 gene on hair follicle organoids 1. Establishment of a hair follicle organoid model: (1) Skin tissue acquisition and predigestion ①Sampling and Pretreatment: Take 0-day-old mice and quickly peel off the skin from their backs with surgical scissors. Wash the skin in pre-cooled PBS containing 1% penicillin-streptomycin on ice.

[0045] ②Removal of subcutaneous tissue: Lay the skin tissue with the dermis side up in a sterile culture dish. Fix the edges of the tissue with forceps, and carefully and thoroughly scrape and cut away the subcutaneous fat tissue and some loose fascia using surgical scissors or a scraper until the surface of the dermis is relatively smooth.

[0046] ③ Dissociation: Transfer the cleaned skin tissue to a 1.5 mL centrifuge tube, add sufficient TrypLEExpress enzyme solution to ensure complete tissue immersion. Cap the tube and place it in a 4°C refrigerator for overnight low-temperature digestion for at least 8 hours. This step aims to gently dissociate the connection between the epidermis and dermis.

[0047] (2) Separation of dermis and epidermis ①Terminate digestion and wash: Remove the centrifuge tube from the 4°C freezer. Use clean tweezers to remove the tissue from the centrifuge tube and place it in a culture dish containing 1% penicillin-streptomycin PBS. Gently shake to wash the tissue. Repeat this step 4 times.

[0048] ② Mechanical separation: Transfer the cleansed skin tissue to a new culture dish (keep it moist with PBS containing a small amount of 1% penicillin-streptomycin). Lay the skin tissue flat with the epidermal side facing up. Firmly grasp one corner of the tissue with fine forceps, and fix the skin tissue with another pair of forceps or a curette. Gently and continuously tear or scrape it to one side horizontally. Ideally, the translucent epidermis will separate completely from the underlying opaque, thicker dermis.

[0049] ③ Tissue aliquoting: Place the separated epidermal and dermal tissues into two new, labeled 1.5 mL centrifuge tubes respectively.

[0050] (3) Enzymatic digestion of dermal and epidermal cells ① Epidermal tissue digestion: Add an appropriate amount (about 1 mL) of 0.25% trypsin solution to a centrifuge tube containing epidermal tissue.

[0051] ② Dermal tissue digestion: Add an appropriate amount (about 1 mL) of 0.1% collagenase I solution to a centrifuge tube containing dermal tissue.

[0052] ③ Incubation and Digestion: Tightly cap the centrifuge tubes and seal them with sealing film to ensure a sterile environment and prevent reagent evaporation or leakage during the water bath. Place the centrifuge tubes in a 37°C water bath for 80 minutes. During digestion, remove the centrifuge tubes from the water bath every 10 minutes and gently invert or shake them 5-10 times to ensure sufficient contact between the enzyme solution and the tissue block, improving digestion efficiency. However, avoid vigorous shaking that could damage cells.

[0053] (4) Preparation, filtration and counting of cell suspensions ① Epidermal cell treatment: a. Termination of digestion: After digestion, add an equal or twice the volume of complete culture medium (containing serum to neutralize trypsin) to the epidermal digestion tube, and gently pipette the tissue block dozens of times with a 1 mL pipette until the tissue block is basically dispersed and the solution becomes turbid.

[0054] b. Cell filtration: Place a 40 μm cell filter into the opening of a new 50 mL centrifuge tube. Transfer all the cell suspension obtained in step a to the top of the filter and allow it to filter naturally. Rinse the original 1.5 mL centrifuge tube twice with a small amount of fresh culture medium, and add the rinse solution to the filter to recover any remaining cells.

[0055] c. Cell collection: After filtration, centrifuge the filtrate in a 50 mL centrifuge tube at 1200 rpm for 4 minutes.

[0056] d. Cell resuspension and counting: Carefully discard the supernatant, avoiding touching cell clumps at the bottom of the tube. Gently resuspend the cells in 30 μL of complete culture medium. Take 1 μL of the cell suspension, dilute with 9 μL of PBS, add 10 μL of trypan blue solution, mix well, and count the cells using a cell counting chamber to calculate the original cell density.

[0057] ② Dermal cell treatment: a. Washing: After dermal tissue digestion, the tissue blocks will appear very loose and the solution viscous to the naked eye. Centrifuge the tube at 1200 rpm for 4 minutes. Carefully discard about half of the supernatant, retaining the remaining liquid and incompletely digested tissue blocks. Add 500 μL of HBSS to the tube, gently pipette to wash away cell clumps and tissue blocks, and centrifuge again at 1200 rpm for 4 minutes. Repeat this washing step 3 times to remove residual collagenase and cell debris.

[0058] b. Cell filtration: After the final centrifugation, discard the supernatant and resuspend the cell clumps with an appropriate amount of HBSS. Following the same steps as for epidermal cell processing, filter the cell suspension into a new 50 mL centrifuge tube using a 40 μm filter and rinse the original tube with HBSS.

[0059] c. Washing and Counting: Centrifuge the filtered cell suspension at 1200 rpm for 4 minutes. Discard the supernatant and repeat the washing and centrifugation twice with HBSS. Resuspend the cell clumps in 500 μL of complete culture medium. Take 1 μL of the cell suspension, dilute it with 9 μL of PBS, add 10 μL of trypan blue staining solution, mix well, and count the cells using a cell counting chamber to calculate the original cell density.

[0060] (5) Cell mixing and inoculation culture ① Proportional Mixing: Based on the counting results and according to the experimental design, calculate and aspirate the corresponding volumes of the two cell suspensions at a ratio of 1:9 for dermal cells to epidermal cells, and add them to a new centrifuge tube. Adjust the total volume and final density to the target using an appropriate amount of complete culture medium.

[0061] ② Mix evenly: Gently pipette the mixture 10 times to ensure even cell distribution and avoid creating air bubbles.

[0062] ③ Seeding: Seed the mixed cell suspension into low-adsorption 96-well plates, adding the recommended volume of 100 μL per well. The low-adsorption surface prevents premature cell adhesion and promotes cell aggregation into spheres, simulating a three-dimensional growth environment.

[0063] ④ Culture: Smoothly transfer the 96-well plate into a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Observe cell aggregation under a microscope 48 hours after inoculation. Carefully aspirate about half of the old culture medium from the wells using a pipette, avoiding aspirating cell clumps, and then slowly add an equal volume of pre-warmed fresh complete culture medium. Subsequently, perform half-volume medium replacement periodically according to the cell growth status.

[0064] 2. Immunofluorescence staining of hair follicle organoids: ① Sampling: Remove the cell culture plate from the incubator and carefully aspirate or pour off the culture medium.

[0065] ② Washing: Gently add pre-warmed PBS (37°C) along the sidewall of the plate and gently agitate to wash the cells, removing residual culture medium and dead cells. Repeat this step twice.

[0066] ③ Fixation: Add sufficient 4% paraformaldehyde to ensure complete coverage of the sample. Fix at room temperature for 15-20 minutes, or at 4°C overnight.

[0067] ④ Washing: Discard the fixative and wash the sample three times with PBS, immersing for 5 minutes each time, on a shaker (low speed) to thoroughly wash away the fixative.

[0068] ⑤ Permeabilization: Add PBST and incubate at room temperature for 10-15 minutes. This step dissolves cell membrane lipids, allowing antibodies to enter the cell and bind to antigens.

[0069] ⑥ Washing: Wash once with PBS for 5 minutes.

[0070] ⑦ Blocking: Aspirate all liquid, add sufficient blocking solution to ensure complete coverage of the sample. Incubate at room temperature for 30 minutes.

[0071] ⑧ Primary antibody incubation: Discard the blocking solution; no washing is required. Dilute the primary antibody directly with antibody dilution buffer at the ratio recommended in the instructions (usually 1:50 to 1:1000) and add it dropwise onto the sample. Cover with sealing film to prevent evaporation and place in a light-proof, humidified chamber. Incubate overnight at 4°C.

[0072] 9. Wash primary antibody: Recover the primary antibody (can be reused 2-3 times). Wash the sample 3 times with PBS (or PBST) containing 1% BSA, 5 minutes each time, on a shaker at low speed.

[0073] ⑩ Secondary antibody incubation: Dilute the fluorescently labeled secondary antibody with secondary antibody dilution buffer or blocking buffer (usually 1:200 to 1:1000). Add the secondary antibody to the sample and cover with sealing film. Place in a light-proof, humidified chamber and incubate at 37°C for 1 hour.

[0074] Washing secondary antibody: Wash 3 times with PBS in the dark, 5 minutes each time, to completely remove unbound secondary antibody.

[0075] Counterstaining cell nuclei: Dilute Hoechst 33342 stock solution to working concentration (5 μg / ml) with PBS. Add Hoechst 33342 solution and incubate at room temperature in the dark for 5-10 minutes.

[0076] Washing: Wash with PBS 2-3 times in the dark, 5 minutes each time, to remove excess Hoechst33342.

[0077] Mounting: Transfer the hair follicle organoids onto a glass slide, add one drop of Vectashield (anti-quenching agent) to the slide, and gently place a coverslip on the mounting medium with tweezers, avoiding air bubbles. Observe under a fluorescence microscope.

[0078] This invention establishes and optimizes a reproducible system for isolating mouse skin cells and culturing three-dimensional hair follicle organoids. Using this system, we demonstrated that overexpression of the SERPINE1 gene in the organoid culture system significantly improves the formation efficiency and quality of hair follicle organoids. Specifically, compared with the control group, the cell aggregates in the SERPINE1 gene overexpression group showed typical hair follicle-like structures earlier, exhibiting a superior morphogenesis trend. Figure 5 Further immunofluorescence analysis showed that the expression level of the cell proliferation marker KI67 was significantly upregulated in hair follicle organoids overexpressing the SERPINE1 gene. Figure 6 The study revealed that the SERPINE1 gene drives organoid development and growth by promoting the proliferative activity of hair follicle-associated cells.

[0079] In summary, this invention not only reproduces and supports the core conclusion that the SERPINE1 gene promotes hair follicle regeneration at the in vitro level, but more importantly, it successfully establishes the SERPINE1 gene as a key factor capable of efficiently optimizing hair follicle organoid culture systems. This discovery provides a novel, higher-performance model construction strategy for in vitro studies of hair follicle development, cycle regulation, and high-throughput drug screening. It bridges the key gap between gene function discovery (in vivo model validation) and efficient in vitro research and application (organoid models), greatly enhancing the practical value and translational potential of this invention in basic research on hair follicle biology, regenerative medicine, and hair-related product development (such as active ingredient screening).

[0080] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The application of the SERPINE1 gene, its encoded protein, or its active derivatives as active ingredients in the preparation of products that promote hair growth and / or hair follicle regeneration, treat hair loss, and in hair follicle organoid models.

2. The application according to claim 1, characterized in that, The products include any one or a combination of cosmetics, skin care products, pharmaceuticals, or medical devices.

3. The application according to claim 1, characterized in that, The medical device is a microneedle, a microneedle array, or a biodegradable implantable material.

4. The application according to claim 1, characterized in that, The SERPINE1 gene is delivered via adenovirus, lentivirus, adeno-associated virus, or retroviral vector.

5. The application according to any one of claims 1-4, characterized in that, The drug or medical device is used to promote the transition of hair follicles from the resting phase to the growth phase, and / or accelerate the hair follicle cycle.

6. The application according to any one of claims 1-4, characterized in that, The drug or medical device is used to prevent or treat androgenetic alopecia, alopecia areata, or telogen effluvium.

7. The application according to any one of claims 1-4, characterized in that, Cosmetics and skincare products are used to promote hair growth or improve thinning hair.

8. The application according to claim 1, characterized in that, The protein encoded by SERPINE1 or its active derivatives promote hair growth or hair follicle regeneration by regulating the PI3K / AKT signaling pathway.

9. The use of a cell containing the SERPINE1 gene or its encoded protein as described in claim 1 in the preparation of a hair growth promoting agent, characterized in that, The cells stably express or overexpress the SERPINE1 gene or its active derivatives.

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