Exosome for treating androgenetic alopecia and application thereof

Abstract

The invention discloses an exosome for treating androgenetic alopecia and application thereof, and belongs to the technical field of biomedicine. The exosome is derived from a human hair papilla cell of overexpression LINC01609. The research result shows that the LINC01609 is obviously reduced in the hair papilla cells of the AGA patient; the engineered exosome obtained by transfecting the overexpression vector can significantly enhance the proliferative activity of hair follicle stem cells (HFSCs). Animal experiments show that the exosome provided by the invention can effectively reverse hair follicle microminiaturization induced by dihydrotestosterone (DHT), so that the hair coverage rate is increased to 92.5% or above, and the diameter of the hair follicle is increased by about 47.6%. Therefore, the invention provides a safe and efficient novel biological means for precise treatment of AGA, and has wide clinical application prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to an exosome for treating androgenetic alopecia and its application. Background Technology

[0002] Androgenetic alopecia (AGA) is the most common progressive hair loss disease in clinical practice, mainly influenced by genetic factors and androgen levels. Its core pathology lies in the dysregulation of the hair follicle microenvironment: under the continuous stimulation of dihydrotestosterone (DHT), the paracrine function of dermal papilla cells (DPCs) becomes disordered, leading to weakened secretion of hair growth-promoting signals (such as Wnt3a, FGF7, VEGF, etc.), ultimately resulting in hair follicle miniaturization and an abnormally shortened growth cycle.

[0003] Currently, FDA-approved treatments mainly include topical minoxidil and oral finasteride. However, minoxidil only improves local blood supply by dilating blood vessels and cannot reverse the intrinsic biological functional damage to hair follicle stem cells, and it has the drawback of relapse after discontinuation. Although finasteride can inhibit DHT production, its systemic use causes side effects such as sexual dysfunction, which severely limits long-term patient compliance.

[0004] In recent years, extracellular vesicles, especially exosomes, have become a research hotspot in the biomedical field due to their excellent biocompatibility, extremely low immunogenicity, and ability to cross the skin's biological barrier. As natural nanocarriers for intercellular communication, exosomes' core advantage lies in their ability to effectively encapsulate and protect bioactive molecules such as nucleic acids and proteins from degradation by various enzymes in the extracellular environment, and precisely deliver these molecules to recipient cells, mediating complex biological functions. However, in the pathological environment of androgenetic alopecia (AGA), exosomes secreted by damaged dermal papilla cells exhibit a severe "lack of active ingredients," resulting in a loss of their intended activating effect on downstream hair follicle stem cells (HFSCs), a significant reason for the irreversible nature of hair loss. Although directly using exosomes from normal sources has some hair regrowth-inducing activity, the concentration of naturally occurring hair regrowth-promoting molecules is often insufficient to produce significant clinical therapeutic effects, failing to achieve the expected therapeutic efficacy. Therefore, how to construct highly active engineered exosome preparations to provide an efficient, safe, and precise treatment for androgenetic alopecia is a key technical problem that urgently needs to be solved. Summary of the Invention

[0005] The purpose of this invention is to provide an exosome preparation for treating androgenetic alopecia and its application, thereby providing a highly effective treatment for androgenetic alopecia.

[0006] To achieve the above objectives, the present invention provides the following technical solution: First, the present invention provides an exosome for treating androgenetic alopecia, wherein the exosome is derived from human hair papilla cells overexpressing LINC01609; The transcript sequence of LINC01609 is shown in SEQ ID NO:2.

[0007] Preferably, the human dermal papilla cells are dermal papilla cells derived from normal individuals.

[0008] Preferably, the androgenetic alopecia is induced by dihydrotestosterone.

[0009] Preferably, the exosomes achieve their therapeutic effect on androgenetic alopecia by promoting the proliferation of hair follicle stem cells.

[0010] Preferably, the method for preparing the exosomes is as follows: (1) The transcript sequence of LINC01609 was amplified using LINC01609 overexpression primers and cloned into a recombinant plasmid to construct the LINC01609 overexpression vector; (2) The LINC01609 overexpression vector was transfected into human dermal papilla cells and cultured to obtain human dermal papilla cells overexpressing LINC01609; (3) After culturing the human hair papilla cells overexpressing LINC01609 in serum-free medium, the culture supernatant was collected and purified by differential centrifugation combined with ultracentrifugation to obtain the exosomes.

[0011] Preferably, the sequences of the LINC01609 overexpression primers are shown in SEQ ID NO:16 and SEQ ID NO:17; The recombinant plasmid is pcDNA3.1 plasmid.

[0012] Secondly, this invention provides the application of the above-mentioned exosomes in the preparation of drugs for treating androgenetic alopecia.

[0013] Preferably, in every 100 μL of the drug, the content of the exosomes is greater than or equal to 100 μg, and the androgenetic alopecia is induced by dihydrotestosterone.

[0014] Preferably, the content of exosomes in each 100 μL of the drug is 100 μg.

[0015] Furthermore, the present invention provides a medicament for treating androgenetic alopecia, characterized in that the medicament is composed of the aforementioned exosomes and a pharmaceutically acceptable matrix.

[0016] Preferably, the androgenetic alopecia is hair loss caused by dihydrotestosterone; The pharmaceutically acceptable matrix is ​​phosphate buffer; The content of exosomes in each 100 μL of the drug is greater than or equal to 100 μg.

[0017] Preferably, the content of exosomes in each 100 μL of the drug is 100 μg.

[0018] The beneficial effects of this invention are as follows: This invention uses genetic engineering to transfect a LINC01609 overexpression vector into normal human dermal papilla cells (Normal-hDPCs), constructing engineered cells that stably overexpress LINC01609, and then isolating and purifying engineered exosomes (Exos-LINC01609) from them.

[0019] Compared to the standard empty vector control exosomes (Exos-Vector), this invention significantly enhances the content and bioactivity of active ingredients in exosomes by overexpressing LINC01609. In vitro experiments demonstrated that Exos-LINC01609 significantly promotes the proliferation of human hair follicle stem cells (HFSCs) more efficiently than standard exosomes, inducing approximately 1.9 times higher proportion of EdU-positive cells in HFSCs compared to the Exos-Vector group, thereby effectively improving dihydrotestosterone-induced hair loss in mice.

[0020] Further in vivo experiments showed that Exos-LINC01609 prepared in this invention can effectively reverse dihydrotestosterone (DHT)-induced hair follicle miniaturization, significantly increase hair follicle diameter, and thus improve the pathological characteristics of androgenetic alopecia. Attached Figure Description

[0021] Figure 1 A graph showing the differential expression of different non-coding RNAs in human dermal papilla cells (hDPCs) from normal individuals and AGA patients; in, Figure 1 a is a statistical graph showing the relative expression levels of LINC01124, LINC02381, and LINC00665; Figure 1 b is a statistical graph showing the relative expression levels of LINC01609 and lncRNA SNHG8; Figure 2The figure shows the overexpression efficiency of AGA-hDPCs after transfection with recombinant plasmids pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8. Figure 3 The figure shows the effect of different groups of hDPCs conditioned medium (CM) on the proliferation capacity of human hair follicle stem cells (HFSCs). Figure 4 Characterization results of exosomes Exos-Vector and Exos-LINC01609 prepared in this invention; in, Figure 4 Image a shows the morphological observation under a transmission electron microscope (TEM). Figure 4 b is a graph showing the results of nanoparticle tracking analysis (NTA); Figure 5 The figure shows the effect of the exosomes Exos-LINC01609 prepared in this invention on the proliferation of HFSCs. Figure 6 The image shows the effect of hair growth on the back of mice in each treatment group after 21 days (a) and the statistical results (b). Figure 7 The figure shows the average diameter of hair follicles in mice of each treatment group after 21 days. Detailed Implementation

[0022] The present invention will be specifically described below through embodiments. It should be noted that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above description.

[0023] Example 1 The steps for obtaining human dermal papilla cells are as follows: Normal group: Occipital scalp of 5 healthy male volunteers (aged 25–35 years); AGA group: 5 male patients with androgenetic alopecia (Norwood classification III–V, age 25–35 years) with bald scalp.

[0024] (1) The scalp tissue of the normal group and the AGA group was washed in DMEM containing 2% antifungal agent ABAM for 10 minutes, and then transferred to DMEM containing 1% ABAM. Excess fat and connective tissue were trimmed with scissors to expose the lower segment of the hair follicle.

[0025] (2) Fix the hair follicle with tweezers, cut it above the matrix, separate the terminal bulb containing the hair papilla, and transfer it into a small droplet containing DMEM + 1% ABAM.

[0026] (3) Use the left hand microneedle to fix the left side of the cylindrical incision, and the right hand microneedle to insert from the bottom of the round and push the inverted structure to expose the hair matrix and hair papilla. (4) Gently peel off the attached matrix cells and brush off the residual matrix with a microneedle; transfer the inverted structure to a large collection droplet and repeat the operation to collect multiple dermal papilla structures.

[0027] (5) Use a microneedle to cut the hair papilla from the connective tissue sheath and transfer it to a 35 mm culture dish containing 2.5 mL DMEM + 20% FBS + 1% ABAM and disperse it evenly.

[0028] (6) Gently scratch the dermal papilla with a needle to make it adhere to the bottom of the dish. Incubate in a 37 ℃, 5% CO2 incubator for 10 days. Then, replace the medium with 10% FBS + 1% penicillin-streptomycin at a ratio of 1:2 to obtain normal human dermal papilla cells (Normal-hDPCs) and human dermal papilla cells derived from AGA patients (AGA-hDPCs).

[0029] Example 2 This embodiment aims to compare the expression differences of five long non-coding RNAs (LINC01124, LINC01609, LINC02381, SNHG8, and LINC00665) selected in this invention between human primary dermal papilla cells derived from the non-bald area of ​​the occipital region of normal volunteers (normal group) and human primary dermal papilla cells derived from the bald area of ​​clinically diagnosed androgenetic alopecia (AGA group) patients (AGA group) using the qRT-PCR method, thereby providing a basis for subsequent treatment of androgenetic alopecia.

[0030] (1) Select normal human dermal papilla cells and AGA human dermal papilla cells with good morphology and positive alkaline phosphatase (ALP) activity from the P3 generation and seed them into 6-well culture plates. Set up 3 biological replicates for each cell type.

[0031] (2) When the cells are fused to 80–90%, wash twice with PBS, add 1 mL of TRIzol Reagent to each well, and lyse at room temperature for 5 min.

[0032] (3) Add 0.2 mL of chloroform, shake vigorously for 15 s, let stand at room temperature for 3 min, centrifuge at 4℃ and 12000g for 15 min, and transfer the upper aqueous phase to a new tube.

[0033] (4) Add an equal volume of isopropanol, precipitate at -20℃ for 30 min, centrifuge at 4℃ and 12,000 g for 10 min, and discard the supernatant.

[0034] (5) Wash the precipitate twice with 75% ethanol, dry at room temperature for 10 min, and dissolve the RNA in 30–50 μL of DEPC water.

[0035] (6) After determining the RNA concentration and purity using NanoDrop and verifying RNA integrity by agarose electrophoresis, 1 μg of total RNA was taken and reverse transcribed using the PrimeScript RT reagent Kit (Takara) according to the instructions to synthesize cDNA.

[0036] (7) Prepare reagents according to the following qRT-PCR reaction system: SYBR Green qPCR Master Mix 10 μL, upstream primer (10 μM) 0.4 μL, downstream primer (10 μM) 0.4 μL, cDNA template 2 μL, ddH2O to bring the total to 20 μL; The primer sequences involved are shown below, and the internal reference gene is GAPDH:

[0037] Perform the PCR reaction according to the following procedure: Pre-denaturation at 95℃ for 30 s; 40 cycles (95℃ for 5 s, 60℃ for 30 s); melting curve analysis (95℃→60℃, 0.5℃ / s).

[0038] (8) Record the Ct value of each gene, calculate ΔCt = Ct(target lncRNA) - Ct(GAPDH), use the average ΔCt of the normal group as a control, calculate ΔΔCt = ΔCt(AGA group) - ΔCt(normal group), and finally calculate the relative expression = 2^(-ΔΔCt).

[0039] The relative expression levels of LINC01124, LINC02381, and LINC00665 are as follows: Figure 1 As shown in Figure a, the relative expression levels of LINC01609 and lncRNA SNHG8 are as follows: Figure 1 As shown in b.

[0040] from Figure 1 The results showed that the relative expression levels of LINC01124, LINC02381, and LINC00665 in human primary dermal papilla cells (hDPCs) from normal individuals and patients with androgenetic alopecia (AGA) did not differ significantly between the AGA and normal groups (P>0.05). This suggests that the expression of these three lncRNAs may be relatively stable under AGA pathological conditions and may not be involved in disease regulation.

[0041] However, the relative expression level of LINC01609 in the AGA group was significantly reduced to 0.27±0.06 (downregulated by approximately 73% compared to the normal group, P<0.01), and the relative expression level of lncRNA SNHG8 was also significantly reduced to 0.42±0.05 (downregulated by approximately 58% compared to the normal group, P<0.01). These data indicate that LINC01609 and lncRNA SNHG8 exhibit a significant downregulated trend in hDPCs derived from AGA patients, suggesting that they may be involved in the development and progression of androgenetic alopecia and are potential therapeutic targets.

[0042] Example 3 Vectors for LINC01609 and lncRNA SNHG8 were constructed and their expression effects were examined. (1) Based on the transcript sequence of LINC01609 and the pcDNA3.1 vector, the following primer sequences were designed: Upstream primer sequence: LINC01609-F: 5'-GGGGGTACCGCACTGGGGGAAAGGGAGAGTATC-3', SEQ ID NO: 16; Downstream primer sequence: LINC01609-R: 5'-GGGCTCGAGTGACTCCGGTCCCCCATGCAATTTC-3', SEQ ID NO: 17.

[0043] (2) Based on the transcript sequence of lncRNA SNHG8 and the pcDNA3.1 vector, the following primer sequences were designed: Upstream primer sequence: SNHG8-F: 5'-GGGGGTACCCACATTCGGGAAGCGTCGGGATTA-3', SEQ ID NO: 18; Downstream primer sequence: SNHG8-R: 5'-GGGCTCGAGTTAGCCACATGAATTAAGCA-3', SEQ ID NO: 19.

[0044] (3) Normal Using hDPCs cDNA as a template, PCR amplification was performed using the primers described above to obtain the target fragments LINC01609 and SNHG8. The PCR products were detected by agarose gel electrophoresis and then purified by gel extraction.

[0045] (4) The purified PCR products were double-digested with KpnI and XhoI respectively; the pcDNA3.1 vector was also digested with the same enzymes. After the digested products were separated by agarose gel electrophoresis, the corresponding target bands and linearized vector fragments were recovered.

[0046] (5) The LINC01609 or lncRNA-SNHG8 insert fragments purified by enzyme digestion are ligated into the linearized pcDNA3.1 vector to obtain recombinant plasmids pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8, which are then sequenced for verification and ready for use.

[0047] (6) AGA human dermal papilla cells were seeded into 6-well plates at a density of 2×10^5 cells / well and cultured in an incubator until 70-80% confluence.

[0048] (7) Before transfection, replace the culture medium with serum-free Opti-MEM and perform transfection according to the following system: 2.5 μg of pcDNA3.1-LINC01609 or pcDNA3.1-SNHG8 plasmid, 5 μL of Lipofectamine 3000, and Opti-MEM to 250 μL. After incubating at room temperature for 20 min, add the plasmid to the cells. The control group is transfected with empty vector pcDNA3.1-Vector. Each group has 3 replicates.

[0049] (8) Six hours after transfection, the culture medium was replaced with complete medium and cultured for another 48 hours. The expression of LINC01609 and SNHG8 was determined according to the method in Example 2.

[0050] Figure 2 The results showed that, compared with the pcDNA3.1-Vector group, the expression level of LINC01609 in the pcDNA3.1-LINC01609 transfection group was significantly upregulated, and the expression level of lncRNA-SNHG8 in the pcDNA3.1-SNHG8 transfection group was also significantly increased. This indicates that both overexpression vectors (pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8) constructed in this study can be effectively overexpressed in AGA human dermal papilla cells.

[0051] Example 4 Effects of conditioned medium on the proliferation of hair follicle stem cells (HFSCs) (1) The P3 generation AGA hDPCs were seeded in 6-well plates (density 2×10^5 cells / well). When the cells adhered to the wells to 70–80% confluence, pcDNA3.1-Vector, pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8 were transfected with Lipofectamine 3000, respectively. After 6 h of transfection, the medium was replaced with complete medium and cultured for another 48 h.

[0052] (2) 48 hours after transfection, discard the old culture medium, wash gently twice with PBS, replace with serum-free DMEM (2 mL per well), and continue culturing for 24 hours.

[0053] (3) Collect the cell culture supernatant of each group, centrifuge at 1000–2000 g for 10 min at 4°C to remove cell debris and dead cells, and obtain conditioned medium CM. After aliquoting the supernatant, store it at -80°C for later use.

[0054] (4) Human hair follicle stem cells HFSCs (Wuhan Pronosai) were seeded in 96-well plates at a density of 5×10^3 cells / well (6 replicates per group).

[0055] (5) Incubate in a 37°C, 5% CO2 incubator until the cells are completely attached to the wall. Discard the original culture medium, wash once with PBS, and add a 1:1 mixture of the above three groups of culture supernatant and fresh HFSC complete culture medium to each well (the culture medium is Mesenchymal Stem Cell Medium, which is used for all hair follicle stem cells described below).

[0056] (6) After incubating in a 37°C, 5% CO2 incubator for 48 hours, add 100 μL of medium containing 50 μM EdU to each well and continue incubation for 4 hours.

[0057] (7) Discard the culture medium, wash twice with PBS, add 100 μL of 4% paraformaldehyde to each well, and fix at room temperature for 30 min.

[0058] (8) After washing 3 times with PBS, add 50 μL of 2 mg / mL glycine to neutralize for 5 min, then permeate with 0.5% Triton X-100 for 10 min, and wash 3 times with PBS.

[0059] (9) Add Apollo staining solution according to the EdU detection kit instructions, incubate at room temperature in the dark for 30 min, wash 3 times with PBS, add Hoechst 33342 to counterstain cell nuclei, and incubate at room temperature in the dark for 20 min.

[0060] (10) After washing with PBS 3 times, 5 non-overlapping fields of view were randomly selected from each well. The number of EdU positive cells and the total number of cells were counted using ImageJ, and the percentage of EdU positive cells was calculated (%).

[0061] Figure 3 The results showed that, compared with the AGA-Control group, the conditioned medium in the LINC01609 overexpression group significantly increased the EdU positivity rate of HFSCs, indicating that overexpression of LINC01609 in hDPCs can effectively activate the proliferation of HFSCs. However, the conditioned medium in the SNHG8 overexpression group had no significant effect on HFSC proliferation, indicating that SNHG8 does not possess similar potential for promoting hair follicle regeneration in this model.

[0062] Example 5 Detecting the effect of LINC01609 or lncRNA-SNHG8 overexpression on the secretory capacity of human dermal papilla cells (1) The obtained P3 generation Normal-hDPCs and AGA-hDPCs were seeded in 6-well culture plates and cultured until 70-80% confluence.

[0063] (2) The cells were grouped as follows: Normal group: Normal-hDPCs cells, without any treatment, served as a healthy control; AGA group: AGA-hDPCs cells transfected with pcDNA3.1-Vector, serving as a disease model control; AGA + LINC01609 group: AGA-hDPCs cells transfected with pcDNA3.1-LINC01609; AGA + SNHG8 group: AGA-hDPCs cells transfected with pcDNA3.1-SNHG8.

[0064] (3) Following the method in Example 3, pcDNA3.1-Vector, pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8 were transfected with Lipofectamine 3000. After 6 h of transfection, the complete culture medium was replaced and cultured for another 48 h.

[0065] (4) 48 hours after transfection, discard the old culture medium, wash gently twice with PBS, replace with serum-free DMEM (2 mL per well), and continue culturing for 24 hours.

[0066] (5) Collect the cell culture supernatant of each group, centrifuge at 1000–2000 g for 10 min at 4°C to remove cell debris and dead cells, and store the supernatant at -80°C for later use.

[0067] (6) The absorbance of Wnt3a, FGF7 and VEGF in the supernatant was detected using an ELISA kit for Wnt3a, FGF7 and VEGF, and the actual concentration of different grouped samples was calculated according to the standard curve.

[0068] Table 1. Concentrations of Wnt3a, FGF7, and VEGF in the conditioned medium for each group of hDPCs

[0069] Note: *** indicates that P < 0.001 compared to the AGA group.

[0070] As shown in Table 1, compared with the normal group, the concentrations of all three factors in the AGA group were significantly lower (P<0.01). Compared with the AGA group, there were no statistically significant differences in the concentrations of each factor in the AGA + SNHG8 group (P>0.05); the concentrations of Wnt3a, FGF7, and VEGF in the AGA + LINC01609 group were significantly higher (P<0.001), recovering to approximately 85.6%, 84.4%, and 81.5% of the normal group, respectively.

[0071] The above results indicate that under AGA background, the secretion of important growth factors such as Wnt3a, FGF7, and VEGF by hDPCs is significantly reduced, leading to weakened paracrine support for downstream hair follicle stem cells (HFSCs). Overexpression of lncRNA-SNHG8 failed to reverse this phenomenon, while overexpression of LINC01609 significantly upregulated the secretion of these factors, suggesting that LINC01609 may indirectly activate HFSC proliferation and hair follicle regeneration by enhancing the secretory function of hDPCs, thus possessing potential therapeutic value for hair loss.

[0072] Example 6 Detecting the effect of LINC01609 or lncRNA-SNHG8 overexpression on the proliferation of human dermal papilla cells (1) The obtained P3 generation Normal-hDPCs and AGA-hDPCs were seeded in 96-well plates at a density of 5×10^3 cells / well and cultured until the cells were completely adhered.

[0073] (2) The cells were grouped as follows (n=6): Normal group: Normal-hDPCs cells, without any treatment, served as a healthy control; AGA group: AGA-hDPCs cells transfected with pcDNA3.1-Vector, serving as a disease model control; AGA + LINC01609 group: AGA-hDPCs cells transfected with pcDNA3.1-LINC01609; AGA + SNHG8 group: AGA-hDPCs cells transfected with pcDNA3.1-SNHG8.

[0074] (3) Following the method in Example 3, pcDNA3.1-Vector, pcDNA3.1-LINC01609 and pcDNA3.1-SNHG8 were transfected with Lipofectamine 3000. After 6 h of transfection, the complete culture medium was replaced and cultured for another 48 h.

[0075] (4) At 0h and 48h, discard the old culture medium in each well and add 100 μL of fresh complete culture medium + 10 μL of CCK-8 solution. At the same time, set up blank control wells (containing only culture medium + CCK-8, without cells).

[0076] (5) Incubate at 37℃ and 5% CO2 for 2 h, and measure the absorbance of each well at 450 nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader.

[0077] Table 2 Results of hDPC proliferation detection in each group

[0078] Table 2 shows that, compared with the AGA-Vector group, the OD450 values ​​of AGA-hDPCs transfected with LINC01609 or SNHG8 were not significantly different at any time point (P>0.05), indicating that overexpression of LINC01609 and SNHG8 had no significant effect on the proliferation capacity of hDPCs. This result suggests that LINC01609 mainly exerts its hair growth-promoting effect through paracrine mechanisms, rather than directly driving the proliferation of dermal papilla cells.

[0079] Example 7 Isolation, purification and identification of exosomes derived from Normal-hDPCs overexpressing LINC01609 (1) P3 generation Normal-hDPCs were seeded in 10cm culture dishes. When the cell confluence reached 70%-80%, the medium was replaced with Opti-MEM medium without exosome FBS. pcDNA3.1-Vector and pcDNA3.1-LINC01609 were transfected according to the method in Example 3. Six hours after transfection, the medium was replaced with complete medium containing 10% exosome FBS and cultured for another 24 hours.

[0080] (2) Discard the old culture medium and wash gently with PBS 3 times to completely remove residual serum.

[0081] (3) Add serum-free DMEM medium and continue culturing at 37°C and 5% CO2 for 48 hours, then collect the culture supernatant.

[0082] (4) After filtering the obtained culture supernatant through a 0.22 μm filter membrane, perform gradient centrifugation at 4℃: 300g for 10 minutes, 2000g for 20 minutes, and 10000g for 30 minutes.

[0083] (5) Transfer the supernatant to an ultracentrifuge tube and centrifuge at 100,000g for 80 minutes.

[0084] (6) Discard the supernatant, gently resuspend the precipitate with pre-cooled PBS, and centrifuge again at 100,000g for 70 minutes.

[0085] (7) Discard the supernatant, add pre-cooled PBS to resuspend the transparent particle precipitate, and obtain Exos-Vector and Exos-LINC01609.

[0086] from Figure 4 The results of a show that the Exos-Vector and Exos-LINC01609 obtained in this invention have a typical bilayer phospholipid membrane structure, are circular, and conform to the basic morphology of exosomes. From Figure 4 The results in b show that the peak of Exos-Vector is located at approximately 110 nm, with most particles distributed in the 50–200 nm range. The main peak of Exos-LINC01609 is approximately 120 nm, with roughly the same range, exhibiting a single-peak distribution, which conforms to the physicochemical property standards of exosomes. Therefore, this invention successfully constructed and extracted engineered LINC01609 exosomes with stable physical properties and meeting purity standards, which can be used for subsequent germinal function verification and AGA treatment research.

[0087] Example 7 Evaluation of the promoting effect of Exos-LINC01609 on the proliferation of human hair follicle stem cells (HFSCs) (1) Human hair follicle stem cells (HFSCs, Wuhan Pronosai) were seeded in 96-well plates at a density of 5×10^3 cells / well, with 6 replicates per group.

[0088] (2) The cells were cultured in a 37°C, 5% CO2 incubator. After the cells were fully adhered, the original culture medium was discarded, and the cells were washed once with PBS. Then, the treatment solution was added according to the following groups: Control group: Add 100 μL of complete culture medium; Exos-Vector group: Add 100 μL of complete culture medium containing 50 μg / mL Exos-Vector exosomes; Exos-LINC01609 group: Add 100 μL of complete culture medium containing 50 μg / mL Exos-LINC01609 exosomes; (3) After incubating in a 37°C, 5% CO2 incubator for 48 hours, add 100 μL of medium containing 50 μM EdU to each well and continue incubation for 4 hours.

[0089] (4) Discard the culture medium, wash twice with PBS, add 100 μL of 4% paraformaldehyde to each well, and fix at room temperature for 30 min.

[0090] (5) After washing with PBS 3 times, add 50 μL of 2 mg / mL glycine to neutralize for 5 min, then permeate with 0.5% Triton X-100 for 10 min, and wash with PBS 3 times.

[0091] (6) Add Apollo staining solution according to the EdU detection kit instructions, incubate at room temperature in the dark for 30 min, wash 3 times with PBS, add Hoechst 33342 to counterstain cell nuclei, and incubate at room temperature in the dark for 20 min.

[0092] (7) After washing with PBS 3 times, 5 non-overlapping fields of view were randomly selected from each well. The number of EdU positive cells and the total number of cells were counted using ImageJ, and the percentage of EdU positive cells was calculated (%).

[0093] from Figure 5The results showed that the percentage of EdU-positive cells in the control group was 15.5±1.0%, the percentage of EdU-positive cells in the Exos-Vector group was 23.9±1.8%, and the percentage of EdU-positive cells in the Exos-LINC01609 group was 44.85±2.4%.

[0094] Compared with the control group, the EdU positivity rate in the Exos-Vector group was slightly increased, but the difference was relatively mild. The Exos-LINC01609 group significantly increased the proportion of EdU-positive cells in HFSCs, approximately 2.9 times higher than the control group and approximately 1.9 times higher than the Exos-Vector group. These results indicate that exosomes derived from Normal-hDPCs transfected with LINC01609 (Exos-LINC01609) can significantly enhance cell proliferation activity. Therefore, exosomes obtained by transfecting dermal papilla cells with LINC01609 have potential value as a novel strategy for AGA treatment.

[0095] Example 8 In vivo efficacy validation of Exos-LINC01609 in a mouse model of DHT-induced androgenetic alopecia (AGA). (1) Laboratory animals and grouping Six- to eight-week-old SPF-grade male C57BL / 6 mice, weighing 18-22 g, were selected and the experiment began after one week of acclimatization.

[0096] Grouping: Model group: Daily subcutaneous injection of DHT (5 mg / kg) at the back of the neck + local injection of 100 μL PBS in the hair removal area on the back; Exos-Vector group: daily subcutaneous injection of DHT (5 mg / kg) at the back of the neck + local injection of 100 μL Exos-Vector exosomes (containing 100 μg exosomes, prepared with PBS) at multiple points in the hair removal area on the back. Exos-LINC01609 group: daily subcutaneous injection of DHT (5 mg / kg) at the back of the neck + multiple local injections of 100 μL Exos-LINC01609 exosomes (containing 100 μg exosomes, prepared with PBS) at the hair removal area on the back.

[0097] (2) AGA model construction and hair removal The day before the experiment, mechanical hair removal was performed on a 2 cm × 3 cm area on the back of all mice. Afterwards, hair removal cream was applied, and the area was gently massaged for 3-5 minutes before being thoroughly washed with warm water to completely remove any remaining hair until the skin on the back turned a uniform pink color. Throughout the experiment (days 1-21), mice in the model group and the two treatment groups were injected subcutaneously with DHT solution (5 mg / kg body weight) at a fixed time of 9:00–10:00 AM daily to simulate hair follicle miniaturization and hair growth inhibition caused by elevated androgen levels.

[0098] (3) Starting from day 1 of the experiment, mice in the Exos-Vector group and the Exos-LINC01609 group were treated with local multi-point subcutaneous injections in the hair-removed area on their backs, and the drugs were administered once every 2 days (i.e., on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, for a total of 11 administrations). Each administration dose is 100 μg of exosomes (Exos-Vector or Exos-LINC01609), prepared with sterile PBS to a total volume of 100 μL, and injected evenly into the hair removal area at 6 points.

[0099] (4) On the 21st day of treatment, after anesthetizing all mice, the backs of the mice were photographed with a digital camera under fixed lighting, distance and angle. The photos were analyzed using ImageJ software to calculate the percentage of hair coverage in the hair-removed area on the back (hair coverage area / total hair-removed area × 100%).

[0100] (5) The mice were euthanized by cervical dislocation, and skin tissue (about 1.0 cm × 1.0 cm) from the central area of ​​the hairless back was quickly cut off, fixed in 4% paraformaldehyde solution for 48 h, routinely embedded in paraffin, sectioned (5 μm thick), and stained with hematoxylin and eosin (H&E).

[0101] (6) Observe the morphology of hair follicles under an optical microscope, randomly select 10 complete hair follicles from each slice, and use ImageJ software to measure the maximum diameter of the hair follicles (μm).

[0102] from Figure 6The results show that the hair coverage percentage in the model group was 11.6±1.7%, with only a very small number of fine vellus hairs visible on day 21, exhibiting obvious hair loss characteristics. The hair coverage percentage in the Exos-Vector group was 40.4±3.3%, with slow but uneven hair growth. The hair coverage percentage in the Exos-LINC01609 group was 92.5±3.4%, with hair almost completely covering the bald area, and the hair was dark and dense, significantly better than the Exos-Vector group.

[0103] from Figure 7 The results showed that the average diameter of hair follicles in the model group was 20.6±1.5μm, in the Exos-Vector group it was 30.7±2.0μm, and in the Exos-LINC01609 group it was 45.3±2.3μm, an increase of approximately 47.6% compared to the Exos-Vector group. These results indicate that local injection of Exos-LINC01609 can effectively reverse DHT-induced hair follicle miniaturization, promote follicle morphological recovery and maintenance of the growth phase, thereby improving the pathological characteristics of androgenetic alopecia.

Claims

1. An exosome for treating androgenetic alopecia, characterized in that, The exosomes were derived from human dermal papilla cells overexpressing LINC01609; The transcript sequence of LINC01609 is shown in SEQ ID NO:

2.

2. The exosomes according to claim 1, characterized in that, The human dermal papilla cells mentioned are dermal papilla cells derived from normal individuals.

3. The exosomes according to claim 2, characterized in that, The androgenetic alopecia mentioned above is induced by dihydrotestosterone.

4. The exosome according to claim 3, characterized in that, The exosomes achieve their therapeutic effect on androgenetic alopecia by promoting the proliferation of hair follicle stem cells.

5. The exosome according to claim 4, characterized in that, The method for preparing the exosomes is as follows: (1) The transcript sequence of LINC01609 was amplified using LINC01609 overexpression primers and cloned into a recombinant plasmid to construct the LINC01609 overexpression vector; (2) The LINC01609 overexpression vector was transfected into human dermal papilla cells and cultured to obtain human dermal papilla cells overexpressing LINC01609; (3) After culturing the human hair papilla cells overexpressing LINC01609 in serum-free medium, the culture supernatant was collected and purified by differential centrifugation combined with ultracentrifugation to obtain the exosomes.

6. The exosome according to claim 5, characterized in that, The sequences of the LINC01609 overexpression primers are shown in SEQ ID NO:16 and SEQ ID NO:17; The recombinant plasmid is pcDNA3.1 plasmid.

7. The use of exosomes as described in any one of claims 1-6 in the preparation of a drug for treating androgenetic alopecia.

8. The application according to claim 7, characterized in that, In every 100 μL of the drug, the content of the exosomes is greater than or equal to 100 μg, and the androgenetic alopecia is induced by dihydrotestosterone.

9. A drug for treating androgenetic alopecia, characterized in that, The drug comprises exosomes as described in any one of claims 1-6 and a pharmaceutically acceptable matrix.

10. The medicament according to claim 9, characterized in that, The androgenetic alopecia mentioned above is hair loss caused by dihydrotestosterone; The pharmaceutically acceptable matrix is ​​phosphate buffer; The content of exosomes in each 100 μL of the drug is greater than or equal to 100 μg.

Images