Mesenchymal stem cell exosome of overexpressed WNT3a protein as well as preparation method and application of mesenchymal stem cell exosome

Abstract

The invention provides a mesenchymal stem cell exosome for overexpressing WNT3a protein as well as a preparation method and application of the mesenchymal stem cell exosome. The exosome provided by the embodiment of the invention is secreted by the engineered mesenchymal stem cells overexpressing the WNT3a protein, the WNT3a protein exists in the exosome and / or on the membrane, and the WNT3a protein can be efficiently and stably conveyed to the hair papilla cells in a targeted manner by utilizing the natural delivery function of the exosome, so that the effect of treating the hair papilla cells is achieved. Cooperative regulation of the WNT3a protein on multiple factors such as hair papilla cell proliferation, related signal channel activation, angiogenesis and local microinflammation relieving environment is achieved, so that hair follicle regeneration and hair growth are effectively promoted, and a brand-new, efficient and safe strategy is provided for preparing the medicine for treating or preventing alopecia.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a mesenchymal stem cell exosome overexpressing WNT3a protein, its preparation method, and its application. Background Technology

[0002] Hair loss, including androgenetic alopecia (AGA) and alopecia areata (AA), is a prevalent dermatological disease worldwide, severely impacting patients' mental health and quality of life. Currently, medications used to treat hair loss primarily include minoxidil (topical) and finasteride (oral). Minoxidil works by opening potassium channels and promoting local blood circulation, but its efficacy varies from person to person and may cause side effects such as contact dermatitis and hirsutism. Finasteride, as a 5α-reductase inhibitor, slows the hair loss process by lowering dihydrotestosterone (DHT) levels, but it carries the potential risk of causing sexual dysfunction and is not suitable for female patients. Furthermore, while hair transplantation surgery offers direct results, it is expensive, invasive, and cannot stop the continued loss of natural hair.

[0003] Therefore, it is urgent to develop a treatment method that is efficient, safe, and has a novel mechanism of action. Summary of the Invention

[0004] In view of this, in order to solve at least one of the above technical problems, embodiments of this application provide a mesenchymal stem cell exosome that overexpresses WNT3a protein.

[0005] Additionally, this application also provides a method for preparing the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein, a pharmaceutical composition, and the use of the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein and the pharmaceutical composition.

[0006] In a first aspect, embodiments of this application provide a mesenchymal stem cell exosome overexpressing the WNT3a protein, the exosome being secreted by engineered mesenchymal stem cells overexpressing the WNT3a protein, the exosome containing the WNT3a protein on its interior and / or membrane.

[0007] Secondly, this application provides a method for preparing mesenchymal stem cell exosomes overexpressing WNT3a protein, comprising: constructing an expression vector containing the coding sequence of the WNT3a gene; introducing the expression vector into mesenchymal stem cells to obtain engineered mesenchymal stem cells overexpressing WNT3a protein; and culturing the engineered mesenchymal stem cells, collecting the supernatant, and separating and purifying the exosomes overexpressing WNT3a protein, wherein the exosomes contain WNT3a protein on their interior and / or membrane.

[0008] Based on the second aspect, in some possible embodiments, the expression vector is a lentiviral vector, an adeno-associated virus vector, or a plasmid.

[0009] Based on the second aspect, in some possible embodiments, the separation and purification steps include: purification using the EXODUS exosome purification system, separation and purification by at least one of ultracentrifugation, size exclusion chromatography and polymer precipitation.

[0010] Based on the second aspect, in some possible embodiments, the mesenchymal stem cells are derived from at least one of umbilical cord, fat, bone marrow, and dental pulp.

[0011] Thirdly, this application also provides a pharmaceutical composition comprising the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein and a pharmaceutically acceptable carrier or diluent.

[0012] Based on the third aspect, in some possible embodiments, the dosage form of the pharmaceutical composition includes a topical dosage form.

[0013] Based on the third aspect, in some possible embodiments, the topical dosage form includes at least one of gel, cream, solution, spray, and microneedle patch.

[0014] Fourthly, this application also provides the use of the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein or the aforementioned pharmaceutical composition in the preparation of a medicament for promoting hair growth, promoting hair follicle regeneration, treating or preventing hair loss.

[0015] Based on the fourth aspect, in some possible embodiments, the hair loss includes at least one of androgenetic alopecia, alopecia areata, chemotherapy-induced alopecia, or nutritional alopecia.

[0016] Compared to existing technologies, the mesenchymal stem cell exosomes overexpressing WNT3a protein provided in this application are secreted by engineered mesenchymal stem cells overexpressing WNT3a protein. The exosomes are enriched with WNT3a protein inside and / or on their membranes. Utilizing the natural delivery function of exosomes, WNT3a protein can be efficiently, stably, and targetedly delivered to dermal papilla cells. This enables the synergistic regulation of multiple factors by WNT3a protein on dermal papilla cell proliferation, activation of related signaling pathways, angiogenesis, and alleviation of the local micro-inflammatory environment. As a result, it effectively promotes hair follicle regeneration and hair growth, providing a novel, efficient, and safe strategy for preparing drugs for the treatment or prevention of hair loss. Attached Figure Description

[0017] Figure 1 This is a schematic flowchart of a method for preparing exosomes according to an embodiment of this application.

[0018] Figure 2 The exosomes provided in Example 1 of this application Wnt3a The transmission electron microscopy image shows that the exosome has a typical cup-shaped morphology.

[0019] Figure 3 The exosomes provided in Example 1 of this application Wnt3a The NTA analysis diagram shows that the particle size of the exosomes is mainly concentrated around 100 nm.

[0020] Figure 4 The exosomes provided in Example 1 of this application Wnt3a Western blot analysis showed that the exosome Exo Wnt3a The marker proteins CD63, CD9, and overexpressed WNT3a protein.

[0021] Figure 5 This is a diagram showing the effect of an in vitro cell experiment provided in Embodiment 2 of this application, wherein, Figure 3 (A) is the diagram for Exo Wnt3a After processing human dermal papilla cells, cell proliferation was detected using the CCK-8 assay. Figure 3 Figure (B) shows the qPCR detection, indicating Exo Wnt3a The expression of Axin, a gene related to the Wnt-β-catenin signaling channel, was significantly upregulated in the experimental group; Figure 3 Figure (C) shows the qPCR detection, indicating Exo Wnt3a The expression of CCDN1, a gene related to the Wnt-β-catenin signaling channel, was significantly upregulated in the experimental group; Figure 3 Figure (D) shows the ELISA test results, indicating Exo Wnt3a The expression of VEGF, a hair regeneration-related factor, was significantly upregulated in the experimental group; Figure 3 Figure (E) shows the ELISA test results, indicating Exo Wnt3a The expression of the pro-inflammatory factor IL-6 was significantly downregulated in the experimental group.

[0022] Figure 6 Photographs of the back of the C57BL / 6 mouse alopecia model provided in Example 3 of this application under different treatments (day 1, day 7, day 15).

[0023] Figure 7 HE staining results of skin tissue samples taken on day 15 of the mouse alopecia model provided in Example 3 of this application under different treatments.

[0024] Figure 8 The image shows the statistical results of hair follicle length in skin tissue taken on day 15 of the mouse alopecia model under different treatments, as provided in Example 3 of this application.

[0025] Figure 9 This is a statistical result of the number of hair follicles in skin tissue taken on day 15 under different treatments in the mouse hair loss model provided in Example 3 of this application.

[0026] Figure 10 The image shows the statistical results of dermal thickness of skin tissue taken on day 15 under different treatments in the mouse hair loss model provided in Example 3 of this application. Detailed Implementation

[0027] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0029] Hair regeneration is a complex process. The inventors of this application have found that this process involves the joint regulation of multiple factors such as cell proliferation, signaling pathway activation, angiogenesis, and local microinflammatory environment. However, there is currently a lack of effective means to synergistically regulate these multiple key links.

[0030] This application provides a mesenchymal stem cell exosome overexpressing the WNT3a protein. The exosome is secreted by engineered mesenchymal stem cells overexpressing the WNT3a protein, and the WNT3a protein is present internally and / or on the membrane of the exosome. This exosome can efficiently enrich and target the delivery of WNT3a protein to dermal papilla cells, synergistically regulating multiple factors affecting hair regeneration, thereby effectively promoting hair follicle regeneration and hair growth.

[0031] First, hair follicle growth is cyclical, including anagen (growth), catagen (regression), and telogen (resting) phases. The Wnt / β-catenin signaling pathway is the "master switch" pathway regulating morphogenesis during embryonic development and cyclical growth after birth. The WNT3a protein loaded in the exosomes of this application possesses complete biological functions. As a core ligand protein of this pathway, WNT3a can effectively activate the WNT / β-catenin signaling pathway within hair follicle cells. After binding to receptors on the cell membrane, WNT3a protein can prevent the degradation of β-catenin, allowing β-catenin to enter the cell nucleus and subsequently initiate the transcription of a series of downstream target genes (such as Axin and CCND1), ultimately promoting the proliferation and differentiation of dermal papilla cells and driving the hair follicle from the telogen phase into the anagen phase. In addition, WNT3a can also simultaneously regulate key factors such as vascular endothelial growth factor (VEGF) and pro-inflammatory cytokine (IL-6), upregulating VEGF expression and downregulating IL-6 expression, thereby promoting angiogenesis and inhibiting inflammation, creating an ideal microenvironment for hair growth. Moreover, the WNT3a protein is loaded into exosomes secreted by the engineered mesenchymal stem cells, rather than obtaining the WNT3a protein and exosomes separately and then combining the two. Therefore, the WNT3a protein loaded on the exosomes of this application has high biological activity and can promote hair follicle regeneration and hair growth more efficiently.

[0032] However, WNT3a protein is unstable in vivo, easily degraded by proteases, and has an extremely short half-life. Therefore, this application utilizes exosomes secreted by engineered mesenchymal stem cells as carriers to load WNT3a protein. These exosomes, as natural delivery carriers, can be effectively taken up by hair follicle cells, facilitating the direct delivery of high concentrations of WNT3a protein to target cells, improving local bioavailability, and solving the problems of difficult transdermal absorption and easy degradation of WNT3a protein. In particular, when WNT3a protein is present on the membrane of exosomes, it can directly contact target cells, more effectively promoting hair follicle regeneration and hair growth. Meanwhile, exosomes secreted by mesenchymal stem cells have advantages such as low immunogenicity, good biocompatibility, and the ability to avoid the potential tumorigenic risks of cell therapy. Furthermore, the phospholipid bilayer structure of the exosome membrane can form steric hindrance, reducing the enzymatic degradation of WNT3a protein by proteases in body fluids. At the same time, these exosomes can also prevent WNT3a protein from being directly recognized and cleared by the immune system, prolonging its circulating half-life in vivo, maintaining the biological activity of WNT3a protein during its circulation and transport in vivo, and reducing the loss and non-specific release of WNT3a protein during transport. Thus, while achieving therapeutic function through efficient targeted delivery, it significantly reduces potential systemic side effects.

[0033] Animal experiments have further confirmed that the exosomes in this application can effectively accelerate hair regeneration in a hair loss model mouse, and significantly increase the number, length and dermal thickness of hair follicles.

[0034] Compared to existing technologies, the mesenchymal stem cell exosomes overexpressing WNT3a protein provided in this application have the following beneficial effects: 1. High biological activity: WNT3a protein derived from living cells has complete biological functions and can effectively activate the WNT / β-catenin signaling pathway in hair follicle cells.

[0035] 2. Synergistic effect of multiple targets: The exosomes of this application not only activate the core WNT / β-catenin pathway, but also regulate key factors such as VEGF and IL-6, and work synergistically from the two levels of promoting growth and inhibiting inflammation, achieving the therapeutic effect of multi-target synergistic effect that is difficult to achieve with single molecule drugs.

[0036] 3. Highly efficient targeted delivery: Mesenchymal stem cell exosomes serve as natural delivery carriers, enabling hair follicle cells to effectively take up the protein and deliver high concentrations of WNT3a protein directly to target cells, thereby improving local bioavailability and solving the problems of difficult transdermal absorption and easy degradation of recombinant proteins.

[0037] 4. Good safety profile: Mesenchymal stem cell exosomes have low immunogenicity and good biocompatibility, avoiding the potential tumorigenic risks of cell therapy and reducing systemic side effects.

[0038] 5. High efficacy: The exosomes in this application have been rigorously verified through in vitro cell experiments and in vivo animal models, from the molecular, cellular, tissue and whole animal levels, in a comprehensive and multi-level manner, demonstrating that exosomes have the effect of effectively promoting hair regeneration.

[0039] Please see Figure 1 This application provides a method for preparing mesenchymal stem cell exosomes overexpressing WNT3a protein. The method specifically includes the following steps: Step S1: Construct an expression vector containing the coding sequence of the WNT3a gene.

[0040] Using genetic engineering techniques, an expression vector carrying the coding sequence of the WNT3a gene was constructed. The coding sequence of the WNT3a gene, such as Seq ID No. 1, is an artificially optimized coding gene for the Wnt3A protein.

[0041] The expression vector can be a lentiviral vector, an adeno-associated virus vector, or a plasmid. The preferred expression vector is a lentiviral vector, and the target gene (WNT3a) mediated by the lentiviral vector can be expressed in mesenchymal stem cells for a long period of time.

[0042] Step S2: The expression vector is introduced into mesenchymal stem cells to obtain engineered mesenchymal stem cells overexpressing WNT3a protein.

[0043] Specifically, for example, an expression vector can be introduced into mesenchymal stem cells via transfection to obtain engineered mesenchymal stem cells that can stably overexpress the WNT3a protein.

[0044] In some embodiments, the multiplicity of infection (MLI) during transfection can be 5 to 40. This suitable MII is beneficial for balancing high transfection efficiency with good cell viability and state. The MII can be exemplarily 5, 10, 15, 20, 25, 30, 35, 40, or any value within the range of any two of the above values.

[0045] Mesenchymal stem cells can be derived from at least one of various tissues, including umbilical cord, umbilical cord blood, fat, bone marrow, and dental pulp.

[0046] Furthermore, the mesenchymal stem cells to be transfected can be selected from mesenchymal stem cells that have been passaged twice (i.e., P2 generation MSCs). P2 generation MSCs are in a vigorous and uniform growth state, which is conducive to transfection. The method of obtaining P2 generation MSCs can be exemplary, including: isolating and culturing primary human mesenchymal stem cells (P0 generation MSCs) from human umbilical cord tissue; when the P0 generation MSCs reach 90% confluence, digesting them and separating them into new culture dishes for further culture to obtain P1 generation MSCs; passage the P1 generation MSCs once more to obtain P2 generation MSCs.

[0047] In some embodiments, after transfection, the cells are screened using antibiotics such as puromycin or G418 to obtain engineered mesenchymal stem cells that overexpress the WNT3a protein.

[0048] Step S3: Culture engineered mesenchymal stem cells, collect the supernatant, and obtain mesenchymal stem cell exosomes overexpressing WNT3a protein by separation and purification. The exosomes contain WNT3a protein on their interior and / or membrane.

[0049] Specifically, the engineered mesenchymal stem cells described above are cultured, and these engineered mesenchymal stem cells secrete exosomes. These exosomes are present in the supernatant. The serum-free culture supernatant is collected, and then separated and purified to obtain the exosomes secreted by the engineered mesenchymal stem cells. The exosomes contain the WNT3a protein, either internally or on their membrane, or both internally and on their membrane.

[0050] In some embodiments, the separation and purification methods include at least one of purification using the EXODUS exosome purification system, ultracentrifugation, size exclusion chromatography, and polymer precipitation. Further, the separation and purification method preferably uses the EXODUS exosome purification system.

[0051] Traditional exosome extraction methods, such as differential ultracentrifugation and density gradient ultracentrifugation, are time-consuming, labor-intensive, and have low recovery rates. They also suffer from low purity and low yield, limiting the application of exosomes. This application also provides a more efficient, high-purity, and high-yield method for exosome extraction, namely, using the EXODUS exosome purification system to purify exosomes.

[0052] Specifically, this application utilizes the EXODUS device developed by Shenzhen Huixin Biotechnology to purify exosomes from mesenchymal stem cell supernatant. The EXODUS exosome purification system is a novel exosome separation method based on ultrasonic nanofiltration, breaking through the bottleneck of traditional membrane separation technology. It applies a negative pressure oscillation and dual-coupled ultrasonic oscillation system to a nanofiltration chip, rapidly removing free nucleic acids and proteins from the sample and retaining exosomes through nanopores, thus achieving exosome enrichment and purification. The stem cell supernatant sample is pretreated, purified, concentrated, and collected using the EXODUS exosome purification system (e.g., the EXODUS T-2800 large-scale fully automated exosome extraction system) to obtain high-yield, high-purity, and high-activity mesenchymal stem cell exosomes.

[0053] Compared with the prior art, the method for preparing mesenchymal stem cell exosomes overexpressing WNT3a protein provided in this application has the following beneficial effects: 1. An expression vector loaded with the WNT3a gene coding sequence was constructed and introduced into mesenchymal stem cells, enabling them to stably and efficiently secrete exosomes loaded with WNT3a protein. This not only solved the stability and targeting problems of WNT3a protein, but also utilized the biological functions of the exosomes themselves to achieve a remarkable effect of multi-target and synergistic promotion of hair regeneration.

[0054] 2. High controllability of preparation process: The mature EXODUS exosome purification system is used for the separation and purification of exosomes. The production process is stable and controllable, and it is easy to carry out quality control and large-scale production.

[0055] This application also provides a pharmaceutical composition comprising, as described above, exosomes and a pharmaceutically acceptable carrier or diluent.

[0056] In some embodiments, the dosage form of the pharmaceutical composition includes a topical dosage form.

[0057] Furthermore, topical formulations can include gels, creams, solutions, sprays, or microneedle patches, which facilitate the transdermal absorption and targeted delivery of exosomes to hair follicles.

[0058] In some embodiments, the content of exosomes in the pharmaceutical composition may be 1 × 10⁻⁶. 10 ~1×10 11The particle size / mL is beneficial for further enhancing the effect of this drug composition in promoting hair follicle regeneration and hair growth.

[0059] This application also provides the use of the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein or the aforementioned pharmaceutical composition in the preparation of a medicament for promoting hair growth, promoting hair follicle regeneration, treating or preventing hair loss.

[0060] In some embodiments, hair loss includes at least one of androgenetic alopecia, alopecia areata, chemotherapy-induced alopecia, or nutritional alopecia.

[0061] The following specific examples further illustrate the aforementioned mesenchymal stem cell exosomes overexpressing WNT3a protein, their preparation methods, and applications.

[0062] Example 1: Mesenchymal stem cell exosomes overexpressing WNT3a protein (Exo Wnt3a Preparation and identification of ) 1. Construct an expression vector containing the coding sequence of the WNT3a gene: (1) PCR cloning of the target gene: Using conventional methods in the field, upstream and downstream primers were designed based on the above expression cassette, and the upstream, downstream, and template DNA were synthesized by Sangon Biotech Co., Ltd. In a PCR tube, 25 μL of 2×Phanta Max Master Mix (Vazyme), 2 μL of upstream primer, 2 μL of downstream primer, and 100 ng of template DNA were added, and the volume was made up to 50 μL with RNase-free ddH2O. After mixing, the tube was placed in a PCR instrument. The PCR reaction program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ final extension for 5 min; and incubation at 4℃. Product recovery: PCR products were separated by 1% agarose gel electrophoresis, the target band was excised, and purified according to the instructions of the gel recovery kit (Vazyme, DC301-01) to obtain the WNT3a target gene (its coding sequence is shown in Seq ID No.1).

[0063] (2) Enzyme digestion and ligation: The target gene fragment and linearized vector were treated with appropriate restriction endonucleases (such as EcoRI and BamHI). The vector was a lentiviral vector backbone pCDH-CAG-MCS-EF1-Puro. The vector was ligated using DNA ligase to obtain an expression vector containing the coding sequence of the WNT3a gene. The specific steps are as follows: In a PCR tube, add 5 μL of 10×CutSmartBuffer, 1 μL of EcoRI enzyme, 1 μL of BamHI enzyme, 2 μg of vector backbone or 1 μg of PCR-purified target gene fragment. Add RNase-free ddH2O to a final volume of 50 μL, mix well, and place in a PCR instrument at 37℃ for 3 h for enzyme digestion. In a PCR tube, add the digested target fragment and vector, 1 μL of T4 DNA ligase and 1 μL of T4 DNA ligase buffer at a molar ratio of 3:1, add RNase-free ddH2O to a final volume of 10 μL, and incubate at room temperature for 10 min.

[0064] (3) Transformation and positive clones: Take 5 μL of ligation product, add 50 μL of DH5α competent cells, and incubate on ice for 30 min; heat shock at 42℃ for 45 s, then quickly incubate on ice for 2 min; add 250 μL of antibiotic-free LB medium, and culture at 37℃ and 220 rpm for h with shaking; take 100 μL of bacterial culture and spread it on LB solid medium containing ampicillin (100 μg / mL), and incubate upside down at 37℃ for 12-16 h. Positive clones are identified by PCR and enzyme digestion, and then sequenced. The plasmid with correct sequencing is the recombinant lentiviral vector (named pCDH-CAG-WNT3A-Puro).

[0065] (4) Lentiviral Packaging: 293T cells were used as lentiviral packaging vector cells. The specific steps are as follows: 293T cells in logarithmic growth phase were taken, the old culture medium was discarded, the cells were washed with sterile PBS, 0.25% trypsin-EDTA digestion solution was added, and the cells were incubated at 37℃ for 1-2 min. Complete culture medium was added to stop digestion, the cells were gently pipetted to form a single-cell suspension, and the cell density was adjusted to 2×10⁻⁶ cells after counting. 5 Cells / mL. Seed the cell suspension into 6-well plates, adding 2 mL of cell suspension to each well, and gently agitate the plate to distribute the cells evenly. Incubate in a CO2 incubator for 24 h. Observe the cell status before transfection: the confluence should reach 70%~80%, and the cells should be plump and morphologically intact.

[0066] Prepare plasmid dilution buffer: Take a sterile EP tube, add 250 μL of Opti-MEM, then add 8 μg of mixed plasmid (4 μg recombinant vector + 3 μg psPAX2 + 1 μg pMD2.G), gently pipette to mix, and let stand at room temperature for 5 min.

[0067] Prepare the transfection reagent dilution solution: Take another sterile EP tube, add 250 μL of Opti-MEM and 16 μL of Lipofectamine 3000 (the mass ratio of transfection reagent to total plasmid is 2:1), mix gently, and let stand at room temperature for 5 min.

[0068] Formation of plasmid-transfection reagent complex: Slowly add the diluted transfection reagent dropwise to the plasmid dilution solution while gently blowing it through the container, and incubate at room temperature for 20 minutes.

[0069] Transfection of 293T cells: Remove the 6-well plate, discard the old culture medium, wash the cells once with Opti-MEM, and add 1.5 mL of Opti-MEM to each well. Slowly add 500 μL of plasmid-transfection reagent complex to each well, and gently shake the culture plate to distribute the complex evenly. Incubate in a CO2 incubator at 37°C and 5% CO2 for 6–8 h. Discard the Opti-MEM containing the transfection complex, add 2 mL of complete culture medium, and continue culturing for 72 h.

[0070] Virus collection: Gently aspirate the supernatant from the 6-well plate using a pipette and transfer it to a 15 mL centrifuge tube, avoiding cell debris. Transfer the virus supernatant to the centrifuge tube and centrifuge at 3000 rpm for 15 min at 4°C to remove cell debris and precipitate. Filter the supernatant through a 0.45 μm PVDF filter membrane to obtain a clear crude virus solution.

[0071] Viral fluid concentration: Transfer the filtered viral fluid to an ultracentrifuge tube and centrifuge at 100,000×g for 2 hours at 4°C. After centrifugation, discard the supernatant and resuspend the precipitate at the bottom of the tube in 1% sterile PBS to obtain concentrated viral fluid. (TCID) 50 The viral titer is calculated using a method that detects and measures viral load.

[0072] 2. The expression vector was introduced into mesenchymal stem cells to obtain engineered mesenchymal stem cells overexpressing WNT3a protein: (1) Cell passage: When the confluence of human primary mesenchymal stem cells reaches 80%~90%, discard the old culture medium, wash the cells with sterile DPBS, add mild stem cell digestive enzymes, and incubate at 37℃ for 2~3 min. Add serum-free complete culture medium specifically for MSCs to terminate digestion, gently pipette the cells to form a single-cell suspension, count the cells, and adjust the cell density to 5000 cells / cm³. 2 The MSCs were seeded into T175 flasks and passaged to the P1 generation. After culturing at 37°C and 5% CO2 for 3-5 days, when the MSC confluence was approximately 90%, the MSCs were passaged to obtain the P2 generation.

[0073] (2) Lentiviral infection: P2 generation MSCs were infected at a rate of 5000 cells / cm². 2Cells were seeded at a density suitable for stem cell culture into T175 flasks and cultured at 37°C and 5% CO2 for 1 day until confluence reached 30%–40%. In a 50ml centrifuge tube, 25ml of fresh serum-free stem cell culture medium and the prepared lentivirus containing the WNT3a gene coding sequence (MOI = 20) were added. Polybrene was then added to a final concentration of 5μg / mL and gently mixed. The old culture medium was discarded, and 25mL of serum-free stem cell culture medium containing lentivirus was added to each T175 flask. The cells were incubated at 37°C and 5% CO2 for 24 hours. 24 hours after infection, the cells were examined under a microscope: slight shrinkage of cells was normal; if a large number of cells floated, the culture medium should be replaced immediately. The virus-containing culture medium was discarded, and the cells were washed once with sterile PBS. 25mL of fresh culture medium was added to each well, and the cells were cultured for another 48 hours.

[0074] (3) Antibiotic screening of positive cells: 72 hours after infection, remove the T175 flask, discard the old culture medium, and add 25 mL of fresh culture medium containing 2 μg / mL puromycin at the screening concentration to each well. Continue culturing in an incubator, changing the screening medium every 2 days for 7-10 days until the cells no longer die or float. When visible cell clusters appear at the bottom of the T175 flask, screening can be stopped, and engineered mesenchymal stem cells overexpressing WNT3a protein (denoted as MSC-WNT3a) can be obtained.

[0075] 3. Culture the engineered mesenchymal stem cells, collect the supernatant, and separate and purify the mesenchymal stem cell exosomes overexpressing WNT3a protein. The exosomes contain WNT3a protein internally and / or on their membranes. (1) The engineered mesenchymal stem cells overexpressing WNT3a protein were divided into groups of 5000 cells / cm². 2 The cells were seeded into commercially available serum-free mesenchymal stem cell culture medium and cultured at 37°C in a 5% CO2 incubator for 4 days. The cell culture supernatant was then collected. (2) Using the EXODUS T-2800 large-scale fully automated exosome extraction system developed by Shenzhen Huixin Biotechnology, the supernatant sample and chip were loaded, and the purification was started by clicking the settings. The exosomes in the supernatant were separated and purified to obtain Exosomes. Wnt3a The exosome Exo Wnt3a Secreted by engineered mesenchymal stem cells (MSC-WNT3a) that overexpress WNT3a protein, the WNT3a protein is present inside the exosome and / or on its membrane; (3) Untransfected MSCs were cultured simultaneously as a control group, and serum-free culture supernatant was collected. The culture supernatant was purified using the EXODUS T-2800 device developed by Shenzhen Huixin Biotechnology: the supernatant sample was loaded onto the chip, the purification was started by clicking the settings, and purified exosomes were obtained, i.e., exosomes without WNT3a protein loading. Control .

[0076] 4. Exosome identification: (1) Transmission electron microscopy observation: 10 μL of exosomes Exo Wnt3a The suspension was dropped onto a copper grid, negatively stained with phosphotungstic acid, and observed under a transmission electron microscope. The results are as follows: Figure 2 As shown, exosomes Exo Wnt3a The typical double-membrane cup structure is consistent with the morphological characteristics of exosomes.

[0077] (2) NTA (Nanoparticle Tracking Analysis) Particle Size Analysis: Exosomes were diluted with PBS. Wnt3a The sample was analyzed using a nanoparticle tracking analyzer. The results are as follows: Figure 3 As shown, exosomes derived from MSC-WNT3a... Wnt3a The particle size is mainly distributed in the range of 30nm-200nm, with a peak value of around 100nm, which is consistent with the characteristics of exosomes.

[0078] (3) Western Blot: Extraction of exosomes (Exosomes) Wnt3a Total protein and exosomes Control Total protein was analyzed by SDS-PAGE electrophoresis and membrane transfer, and then detected using antibodies against CD9, CD63, CD81, and WNT3a. Results are as follows: Figure 4 As shown, all exosome samples expressed the exosome-specific marker proteins CD9, CD63, and CD81, while only exosomes derived from MSC-WNT3a expressed the specific marker proteins CD9, CD63, and CD81. Wnt3a High expression of WNT3a protein demonstrates successful preparation of Exo Wnt3a .

[0079] Example 2, Exo Wnt3a In vitro functional verification 1. Cell proliferation detection (CCK-8 assay): Human dermal papilla cells were divided into 5 × 10⁻⁶ cells per well. 3 Cells were seeded in 96-well plates and divided into four groups: negative control (NC), dihydrotestosterone (DHT) modeling group, DHT+Exo group, and DHT+Exo group. Control Group, DHT+Exo Wnt3a Groups were set up, with 5 replicates in each group; after human dermal papilla cells adhered to the wells, they were divided into experimental groups according to the experimental groups, except for the negative control group, the DHT modeling group and the experimental group (DHT+Exo)Control Group and DHT+Exo Wnt3a Groups were induced with 100 μM DHT for 24 h and cultured at 37℃ in a 5% CO2 incubator for 24 h. Treatments were administered according to the experimental groups: the negative control group was treated with 10% PBS, the DHT modeling group was treated with 10% PBS, and the DHT+Exo group was treated with... Control The concentration added to the group at 10% volume is 3×10 10 Exo particles / mL Control and DHT+Exo Wnt3a The concentration added to the group at 10% volume is 3×10 10 Exo particles / mL Wnt3a Treatment: After culturing for another 48 hours, 10 μL of CCK-8 solution (Cell Counting Kit-8, used to test the activity of dehydrogenases in cells, reflecting the number of viable cells and metabolic activity) was added to each well. After incubation for 2 hours, the absorbance was measured at 450 nm using a microplate reader. Results are as follows: Figure 5 As shown in Figure A, with DHT modeling group and DHT+Exo Control Compared to the previous group, DHT+Exo Wnt3a The cell proliferation activity of the group was significantly enhanced at 48 h.

[0080] 2. qPCR detection of pathway gene expression: Cells were treated with different conditions as described above for 48 hours to detect cell proliferation. Total RNA was extracted from four groups of cells, reverse transcribed into cDNA, and then analyzed by real-time quantitative PCR. GAPDH was used as an internal control to detect the mRNA expression levels of Axin and CCND1. Results are as follows: Figure 5 China B diagram and Figure 5 As shown in Figure C, DHT+Exo Wnt3a The expression levels of Axin and CCND1 in group A were significantly higher than those in other groups, indicating that the Wnt / β-catenin signaling pathway is blocked by Exo Wnt3a Activation successful.

[0081] 3. ELISA (Enzyme-Linked Immunosorbent Assay) detection of secreted factors: Cells were treated with the different conditions described above for 48 hours to detect cell proliferation. Cell supernatant was collected, and the concentrations of VEGF and IL-6 were measured strictly according to the instructions of the ELISA kits (VEGF ELISA kit was model ml064255 from Shanghai Enzyme-Link Biotechnology Co., Ltd., and IL-6 ELISA kit was model VAL102 from NOVUS). Results are as follows: Figure 5 Chinese D diagram and Figure 5 As shown in Figure E, with DHT modeling group and DHT+ExoControl Compared to the previous group, DHT+Exo Wnt3a The group significantly promoted VEGF secretion while significantly inhibiting IL-6 secretion, demonstrating Exo Wnt3a It has a good dual regulatory function of promoting hair growth and anti-inflammation.

[0082] Example 3, Exo Wnt3a Animal in vivo experiments to verify (1) Animal model establishment: Seven-week-old healthy C57BL / 6 mice were selected. All mice were anesthetized and fixed with isoflurane. The hair on the back of the mice was shaved off with a razor, covering an area of ​​about 2cm × 3cm. Then, depilatory cream was applied to remove the remaining hair. Mice with pink hair in the resting phase were selected for the group and randomly divided into four groups of 6 mice each: negative control group (NC), androgenic alopecia (AGA) modeling group, Exo Control Experimental group, Exo Wnt3a The experimental group was used to establish an animal model. The modeling method was as follows: Except for the negative control group (NC) mice which were injected intraperitoneally with 100 μL of solvent (5% DMSO + 95% corn oil), the model group and experimental group mice were injected intraperitoneally with 100 μL of 10 mg / mL DHT for modeling. The injection was once a day for 15 days, and the day of modeling was recorded as Day 1. (2) Drug administration and observation: According to the grouping, the drug was injected subcutaneously at ten sites in the hair removal area daily, with 50 μl injected at each site, for a total of 500 μl. Specifically, the negative control group received a total of 500 μl of PBS at ten sites, the AGA modeling group received a total of 500 μl of PBS at ten sites, and Exo... Control The experimental group received a total injection of 500 μl at ten sites, with a concentration of 3 × 10⁻⁶. 10 Exo particles / mL Control Exo Wnt3a The experimental group received a total injection of 500 μl at ten sites, with a concentration of 3 × 10⁻⁶. 10 Exo particles / mL Wnt3a For 15 consecutive days, all mice were observed and photographed every two days. Figure 6 As shown, after DHT modeling, Exo Wnt3a The experimental group of mice had the largest area of ​​newly grown hair coverage and the highest density.

[0083] (3) Histological analysis: 24 hours after the last administration, skin tissue from the hair-removed areas of 3 mice in each group was collected, fixed in 10% formalin, and then embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE staining method), scanned panoramically, and quantitatively analyzed for the number of hair follicles, dermal thickness, and hair follicle length. The results are as follows: Figures 7 to 10 As shown, Exo Wnt3a The hair follicle length and dermal thickness in the experimental group were significantly higher than those in the AGA modeling group and the Exo group.Control Experimental group, Exo Wnt3a The number of hair follicles in the experimental group was significantly higher than that in the AGA modeling group, Exo Wnt3a The experimental group showed higher overall performance in promoting hair growth.

[0084] The above experiments fully demonstrate that the exosomes secreted by engineered mesenchymal stem cells overexpressing WNT3a protein provided in this application, and the presence of WNT3a protein inside and / or on the membrane of the exosomes, can effectively promote hair follicle cell proliferation, activate key signaling pathways for hair growth, and exhibit excellent hair growth promotion capabilities in cell and animal models, showing promising clinical application prospects.

[0085] The above description of the embodiments is only for the purpose of helping to understand the method and core idea of ​​this application; in addition, those skilled in the art can make various other corresponding changes and modifications based on the technical concept of this application, and all such changes and modifications should fall within the protection scope of the claims of this application.

Claims

1. A mesenchymal stem cell exosome overexpressing WNT3a protein, characterized in that, The exosomes are secreted by engineered mesenchymal stem cells that overexpress the WNT3a protein, and the WNT3a protein is present on the interior and / or membrane of the exosomes.

2. A method for preparing mesenchymal stem cell exosomes overexpressing WNT3a protein, characterized in that, include: Construct an expression vector containing the coding sequence of the WNT3a gene; The expression vector was introduced into mesenchymal stem cells to obtain engineered mesenchymal stem cells overexpressing WNT3a protein. as well as The engineered mesenchymal stem cells were cultured, and the supernatant was collected. The mesenchymal stem cell exosomes overexpressing WNT3a protein were obtained by separation and purification. The WNT3a protein was present inside the exosomes and / or on their membranes.

3. The preparation method according to claim 2, characterized in that, The expression vector is a lentiviral vector, an adeno-associated virus vector, or a plasmid.

4. The preparation method according to claim 2, characterized in that, The separation and purification steps include: The exosomes were purified using the EXODUS exosome purification system, or separated and purified by at least one of ultracentrifugation, size exclusion chromatography, and polymer precipitation.

5. The preparation method according to claim 2, characterized in that, The mesenchymal stem cells are derived from at least one of the following: umbilical cord, fat, bone marrow, and dental pulp.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises mesenchymal stem cell exosomes overexpressing WNT3a protein as described in claim 1, and a pharmaceutically acceptable carrier or diluent.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage forms of the pharmaceutical composition include topical dosage forms.

8. The pharmaceutical composition according to claim 7, characterized in that, The topical dosage forms include gels, creams, solutions, sprays, or microneedle patches.

9. Use of a mesenchymal stem cell exosome overexpressing WNT3a protein as described in claim 1, or a pharmaceutical composition as described in any one of claims 6 to 8, in the preparation of a medicament for promoting hair growth, promoting hair follicle regeneration, treating or preventing hair loss.

10. The use according to claim 9, characterized in that, The hair loss includes at least one of androgenetic alopecia, alopecia areata, chemotherapy-induced alopecia, or nutritional alopecia.

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