Preparation and application of hair follicle stem cell exosome

Abstract

The invention relates to the technical field of hair follicle stem cell exosomes, in particular to a preparation method of a hair follicle stem cell exosome, which comprises the following steps: S1, separation and culture of hair follicle stem cells: a, collecting a sample, selecting hair follicle tissues of healthy human beings, and selecting hair follicles from a dense hair area; b, separating hair follicle stem cells, namely cutting hair follicle tissues into small sections by adopting a collagenase / trypsin digestion method, digesting to remove non-hair follicle tissues, centrifuging digested cell suspension by adopting a Percoll gradient method, layering, separating out hair follicle stem cell groups, further purifying the hair follicle stem cells by using flow cytometry and magnetic bead sorting, and according to surface markers of the hair follicle stem cells, separating the hair follicle stem cells from the hair follicle stem cells to obtain the hair follicle stem cells. The identification is carried out on CD34 and CD200; c, hair follicle stem cell culture: using a stem cell culture medium to culture hair follicle stem cells. When the technology is used for preparing the exosome of the hair follicle stem cell, damage to exosome vesicles in the preparation process can be reduced, the yield of the exosome is increased, and meanwhile, the target exosome can be accurately obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of hair follicle stem cell exosomes, and in particular to the preparation and application of hair follicle stem cell exosomes. Background Art

[0002] Hair follicle stem cells (HFSCs) are relatively primitive cells in hair follicles. They are mesenchymal stem cells with strong self-renewal and multidirectional differentiation abilities. They are located in the outermost layer of the bulge of the outer root sheath of the hair follicle, close to the attachment point of the arrector pili muscle. Hair follicle stem cells can not only differentiate into hair follicles and effectively treat hair loss, but also participate in the formation of epidermal tissue. At the same time, they have strong immunomodulatory effects, hematopoietic support, and promote tissue repair.

[0003] Exosomes are a type of extracellular vesicles secreted by cells, with a particle size of about 30nm-200nm. They usually carry bioactive molecules such as proteins, mRNA, miRNA and lipids, and can integrate these bioactive molecules into target cells to achieve cell interaction. Therefore, they play an important role in cell signaling. Due to the above characteristics, exosomes have been applied in many fields.

[0004] In the prior art, regarding the preparation method of hair follicle stem cell exosomes, the hair follicle stem cell exosomes have poor preparation and culture stability during the preparation process. The hair follicle stem cell exosomes are easily damaged during the culture preparation process, thereby affecting the proliferation and activity of the cells.

[0005] To this end, the preparation and application of hair follicle stem cell exosomes are proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation and application of hair follicle stem cell exosomes. The preparation of hair follicle stem cell exosomes by the technology of the present invention can reduce the damage to the exosome vesicles during the preparation process, increase the yield of exosomes, and accurately obtain the target exosomes, so as to solve the problems raised in the above background technology.

[0007] To achieve the above object, the present invention provides the following technical solution: a preparation method of hair follicle stem cell exosomes, comprising the following steps:

[0008] S1. Isolation and culture of hair follicle stem cells:

[0009] a. Sample collection: select hair follicle tissue from healthy humans and select hair follicles from hair-dense areas;

[0010] b. Isolation of hair follicle stem cells: using collagenase / trypsin digestion method to cut hair follicle tissue into small segments, digestion to remove non-hair follicle tissue, centrifuging the digested cell suspension through Percoll gradient method, stratifying, separating the hair follicle stem cell population, using flow cytometry and magnetic bead sorting to further purify hair follicle stem cells, and identifying them based on their surface markers, namely CD34 and CD200;

[0011] c. Hair follicle stem cell culture: hair follicle stem cells are cultured using stem cell culture medium, and FGF-7 and EGF growth factors are added to the culture medium to promote cell proliferation and activity;

[0012] S2. Extraction and purification of exosomes:

[0013] Cell culture and collection: hair follicle stem cells were cultured in serum-free medium until 80% to 90% confluence, residual FBS in the cell culture medium was removed to avoid interference from exosomes in the serum, and the hair follicle stem cell culture medium was collected and centrifuged at low speed at 3000 rpm for 10 minutes to remove cell debris and impurities;

[0014] Ultra-high-speed centrifugation is used to extract exosomes. The culture medium after cell removal is centrifuged at high speed, specifically 100,000 x g, for 1 h-2 h. The precipitate is collected. The precipitate is the crude exosome extract. The exosomes are further purified by density gradient centrifugation. Different concentration gradients are made by using 1.13, 1.19, and 1.22 KBr solutions to separate high-purity exosomes.

[0015] For the identification of exosomes, the morphology of exosomes was observed by transmission electron microscopy to confirm that they had a typical nanoscale spherical structure, specifically 50nm-150nm. Nanoparticle tracking analysis was used to determine the particle size distribution and concentration of exosomes. Western Blot was used to detect exosome-specific markers, namely CD9, CD63, TSG101, and Alix. Dynamic light scattering was used to further determine the particle size distribution of exosomes and confirm the purity.

[0016] Preferably, in said S2, the extraction and purification of exosomes, hair follicle stem cells are cultured in serum-free medium and in a hypoxic environment, wherein the hypoxic parameter is 1% to 5% O2 concentration, to stimulate the metabolic activity of the cells and increase the production of exosomes.

[0017] Preferably, in said S2, fiber growth factor and insulin growth factor are added during the extraction and purification of exosomes to promote the proliferation of hair follicle stem cells and enhance the biological activity of exosomes.

[0018] An application of hair follicle stem cell exosomes, including application in hair regeneration and skin repair;

[0019] Among them, the exosomes of hair regeneration follicle stem cells contain many molecules related to hair growth and hair follicle formation, namely TGF-β, Wnt, and IGF, which promote hair growth through the following pathways:

[0020] Stimulate the proliferation and differentiation of hair follicle stem cells: miRNAs in the exosomes of hair follicle stem cells, namely miR-21 and miR-22, regulate the proliferation and differentiation of hair follicle stem cells and activate hair follicle growth;

[0021] Activation of hair growth signaling pathways: Proteins in exosomes, namely Wnt, β-catenin, and miRNA activate hair growth-related signaling pathways;

[0022] Promote angiogenesis and nutrient supply: Hair follicle stem cell exosomes contain molecules that can promote angiogenesis, namely VEGF and HGF.

[0023] Preferably, the skin repair, the role of hair follicle stem cell exosomes in skin damage repair: anti-inflammatory effect: miRNA and protein molecules in hair follicle stem cell exosomes can regulate immune response.

[0024] Preferably, the modification and function enhancement optimization of exosomes are also included, specifically, the surface modification and targeting enhancement of exosomes, the load and function enhancement, and the stability and storage of exosomes.

[0025] Preferably, the exosome surface modification and targeting enhancement are:

[0026] Targeted molecular modification: Target it to specific cells or tissues through surface modification, use antibodies or peptide molecules to modify the surface of exosomes, and specifically target hair follicle stem cells and skin epidermal cells.

[0027] Antibody modification: Antibodies targeting specific surface receptors are attached to the surface of exosomes to enhance the specific recognition of exosomes to target cells.

[0028] Peptide modification: using RGD peptide to enhance the targeting of exosomes to skin cells;

[0029] Surface charge regulation of exosomes: Changing the charge on the surface of exosomes can regulate their affinity to target cells.

[0030] Preferably, the load and function enhancement: Gene load: using exosomes as gene carriers to load specific genes and small interfering RNA.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The preparation of hair follicle stem cell exosomes by the technology of the present invention can reduce the damage to the exosome vesicles during the preparation process, increase the yield of exosomes, and accurately obtain the target exosomes. ;

[0033] 2. The quality and yield of exosomes can be effectively improved by improving culture conditions, increasing production scale, and improving extraction and purification methods. The functionality of exosomes can be enhanced and their application effect in clinical treatment can be improved by surface modification, loading drugs or genes, and increasing stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is a flow chart for preparing hair follicle stem cell exosomes of the present invention;

[0036] Figure 2 This is an application category of hair follicle stem cell exosomes of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] See also Figure 1 to Figure 2 , the present invention provides a technical solution:

[0039] A preparation and application of hair follicle stem cell exosomes, comprising the following steps:

[0040] S1. Isolation and culture of hair follicle stem cells:

[0041] a. Sample collection: select hair follicle tissue from healthy humans and select hair follicles from hair-dense areas;

[0042] b. Isolation of hair follicle stem cells: using collagenase / trypsin digestion method to cut hair follicle tissue into small segments, digestion to remove non-hair follicle tissue, centrifuging the digested cell suspension through Percoll gradient method, stratifying, separating the hair follicle stem cell population, using flow cytometry and magnetic bead sorting to further purify hair follicle stem cells, and identifying them based on their surface markers, namely CD34 and CD200;

[0043] c. Hair follicle stem cell culture: hair follicle stem cells are cultured using stem cell culture medium, and FGF-7 and EGF growth factors are added to the culture medium to promote cell proliferation and activity;

[0044] S2. Extraction and purification of exosomes:

[0045] Cell culture and collection: hair follicle stem cells were cultured in serum-free medium until 80% to 90% confluence, residual FBS in the cell culture medium was removed to avoid interference from exosomes in the serum, and the hair follicle stem cell culture medium was collected and centrifuged at low speed at 3000 rpm for 10 minutes to remove cell debris and impurities;

[0046] Ultra-high-speed centrifugation is used to extract exosomes. The culture medium after cell removal is centrifuged at high speed, specifically 100,000 x g, for 1 h-2 h. The precipitate is collected. The precipitate is the crude exosome extract. The exosomes are further purified by density gradient centrifugation. Different concentration gradients are made by using 1.13, 1.19, and 1.22 KBr solutions to separate high-purity exosomes.

[0047] For the identification of exosomes, the morphology of exosomes was observed by transmission electron microscopy to confirm that they had a typical nanoscale spherical structure, specifically 50nm-150nm. Nanoparticle tracking analysis was used to determine the particle size distribution and concentration of exosomes. Western Blot was used to detect exosome-specific markers, namely CD9, CD63, TSG101, and Alix. Dynamic light scattering was used to further determine the particle size distribution of exosomes and confirm the purity.

[0048] Specifically, in the S2, extraction and purification of exosomes, hair follicle stem cells are cultured in serum-free medium and in a hypoxic environment, wherein the hypoxic parameter is 1% to 5% O2 concentration, which stimulates the metabolic activity of the cells and increases the production of exosomes.

[0049] Specifically, in the S2, fiber growth factor and insulin growth factor are added during the extraction and purification of exosomes to promote the proliferation of hair follicle stem cells and enhance the biological activity of exosomes.

[0050] An application of hair follicle stem cell exosomes, including application in hair regeneration and skin repair;

[0051] Among them, the exosomes of hair regeneration follicle stem cells contain many molecules related to hair growth and hair follicle formation, namely TGF-β, Wnt, and IGF, which promote hair growth through the following pathways:

[0052] Stimulate the proliferation and differentiation of hair follicle stem cells: miRNAs in the exosomes of hair follicle stem cells, namely miR-21 and miR-22, regulate the proliferation and differentiation of hair follicle stem cells and activate hair follicle growth;

[0053] Activate hair growth signaling pathway: The proteins in exosomes, namely Wnt, β-catenin and miRNA, activate the hair growth-related signaling pathway, namely the Wnt / β-catenin pathway, and promote hair follicle regeneration;

[0054] Promote angiogenesis and nutrient supply: Hair follicle stem cell exosomes contain molecules that can promote angiogenesis, namely VEGF and HGF. These molecules can improve the local microenvironment, promote the nutrient supply of hair follicle tissue, and thus promote hair growth.

[0055] Specifically, in the skin repair, hair follicle stem cell exosomes also show significant effects in skin damage repair:

[0056] Anti-inflammatory effect: miRNA, proteins and other molecules in hair follicle stem cell exosomes can regulate the immune response and inhibit the release of inflammatory factors (such as TNF-α, IL-1β), thereby reducing the inflammatory response and accelerating skin healing.

[0057] Promote wound healing: Growth factors in exosomes (such as EGF, HGF, VEGF, etc.) help heal skin wounds and accelerate the wound repair process by promoting the proliferation and migration of keratinocytes and fibroblasts.

[0058] Promote collagen synthesis and tissue regeneration: miRNA in exosomes (such as miR-29, miR-146) can enhance the activity of fibroblasts, promote collagen synthesis, increase skin elasticity, and improve skin tissue structure.

[0059] Specifically, it also includes the modification and function enhancement optimization of exosomes, specifically the surface modification and targeting enhancement of exosomes, the enhancement of loading and function, the stability and storage of exosomes

[0060] Specifically, the exosome surface modification and targeting enhancement:

[0061] Targeted molecule modification: In order to improve the targeting of exosomes, they can be positioned to specific cells or tissues through surface modification. For example, antibodies or peptide molecules can be used to modify the surface of exosomes to specifically target hair follicle stem cells or skin epidermal cells. This can not only improve the targeted therapeutic efficiency of exosomes, but also reduce side effects.

[0062] Antibody modification: Antibodies targeting specific surface receptors (such as CD200 antibodies) can be attached to the surface of exosomes to enhance the specific recognition of exosomes to target cells (such as hair follicle cells).

[0063] Peptide modification: For example, RGD peptide (a short peptide that binds to cell receptors) is used to enhance the targeting of exosomes to skin cells.

[0064] Surface charge regulation of exosomes: Changing the charge on the surface of exosomes (through polymer modification or other chemical methods) can regulate their affinity with target cells. Surface modification with positive or negative charge can help improve the cellular uptake efficiency of exosomes.

[0065] Specifically, the load and function enhancements include:

[0066] In addition to natural bioactive molecules (such as miRNA, mRNA, proteins, lipids, etc.), exosomes can also enhance their functions by loading exogenous molecules:

[0067] Gene loading: Using exosomes as gene carriers to load specific genes or small interfering RNA (siRNA, miRNA), etc., can further enhance the therapeutic effect of exosomes. For example, specific miRNA or gene editing tools (such as CRISPR-Cas9) can be loaded to promote hair follicle regeneration or repair skin.

[0068] Small molecule drug loading: Loading small molecule drugs (such as anti-inflammatory drugs, immunomodulatory drugs) into exosomes to improve the targeting and bioavailability of drugs. This can improve the therapeutic effect while reducing the side effects of drugs.

[0069] Specifically, the stability of the exosomes is a key issue in their clinical application, especially during storage, transportation and application. The following are optimization strategies to improve the stability of exosomes:

[0070] Freeze-drying technology: Freeze-drying technology can effectively improve the stability of exosomes and facilitate long-term storage and transportation. Freeze-dried exosomes can restore their biological activity after reconstitution, so freeze-drying is an important technology for large-scale application of exosomes.

[0071] Carrier protection: Co-encapsulation of exosomes with nanocarriers or liposomes can effectively protect the stability of exosomes during in vitro storage and application, and avoid functional loss caused by environmental factors (such as temperature, pH changes, etc.).

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of hair follicle stem cell exosomes, characterized in that: The steps include: S1. Isolation and culture of hair follicle stem cells: a. Sample collection: select hair follicle tissue from healthy humans and select hair follicles from hair-dense areas; b. Isolation of hair follicle stem cells: using collagenase / trypsin digestion method to cut hair follicle tissue into small segments, digestion to remove non-hair follicle tissue, centrifuging the digested cell suspension through Percoll gradient method, stratifying, separating the hair follicle stem cell population, using flow cytometry and magnetic bead sorting to further purify hair follicle stem cells, and identifying them based on their surface markers, namely CD34 and CD200; c. Hair follicle stem cell culture: hair follicle stem cells are cultured using stem cell culture medium, and FGF-7 and EGF growth factors are added to the culture medium to promote cell proliferation and activity; S2. Extraction and purification of exosomes: Cell culture and collection: hair follicle stem cells were cultured in serum-free medium until 80% to 90% confluence, residual FBS in the cell culture medium was removed to avoid interference from exosomes in the serum, and the hair follicle stem cell culture medium was collected and centrifuged at low speed at 3000 rpm for 10 minutes to remove cell debris and impurities; Ultra-high-speed centrifugation is used to extract exosomes. The culture medium after cell removal is centrifuged at high speed, specifically 100,000 x g, for 1 h-2 h. The precipitate is collected. The precipitate is the crude exosome extract. The exosomes are further purified by density gradient centrifugation. Different concentration gradients are made by using 1.13, 1.19, and 1.22 KBr solutions to separate high-purity exosomes. For the identification of exosomes, the morphology of exosomes was observed by transmission electron microscopy to confirm that they had a typical nanoscale spherical structure, specifically 50nm-150nm. Nanoparticle tracking analysis was used to determine the particle size distribution and concentration of exosomes. Western Blot was used to detect exosome-specific markers, namely CD9, CD63, TSG101, and Alix. Dynamic light scattering was used to further determine the particle size distribution of exosomes and confirm the purity.

2. The preparation of hair follicle stem cell exosomes according to claim 1, characterized in that: In the S2, extraction and purification of exosomes, hair follicle stem cells are cultured in serum-free medium and in a hypoxic environment, wherein the hypoxic parameter is 1% to 5% O2 concentration, which stimulates the metabolic activity of the cells and increases the production of exosomes.

3. The preparation of hair follicle stem cell exosomes according to claim 2, characterized in that: In the S2, fiber growth factor and insulin growth factor are added during the extraction and purification of exosomes.

4. The use of hair follicle stem cell exosomes according to any one of claims 1 to 3, characterized in that: Including applications in hair regeneration and skin repair; Among them, the exosomes of hair regeneration follicle stem cells contain many molecules related to hair growth and hair follicle formation, namely TGF-β, Wnt, and IGF, which promote hair growth through the following pathways: Stimulate the proliferation and differentiation of hair follicle stem cells: miRNAs in the exosomes of hair follicle stem cells, namely miR-21 and miR-22, regulate the proliferation and differentiation of hair follicle stem cells and activate hair follicle growth; Activation of hair growth signaling pathways: Proteins in exosomes, namely Wnt, β-catenin, and miRNA activate hair growth-related signaling pathways; Promote angiogenesis and nutrient supply: Hair follicle stem cell exosomes contain molecules that promote angiogenesis, namely VEGF and HGF.

5. The use of hair follicle stem cell exosomes according to claim 4, characterized in that: The skin repair, the role of hair follicle stem cell exosomes in skin damage repair: Anti-inflammatory effect: miRNA and protein molecules in hair follicle stem cell exosomes can regulate immune response.

6. The preparation of hair follicle stem cell exosomes according to claim 1, characterized in that: It also includes the modification and function enhancement optimization of exosomes, specifically the surface modification and targeting enhancement of exosomes, loading and function enhancement, and the stability and storage of exosomes.

7. The preparation of hair follicle stem cell exosomes according to claim 6, characterized in that: The exosome surface modification and targeting enhancement: Targeted molecular modification: Targeting to specific cells and tissues through surface modification, using antibodies and peptide molecules to modify the surface of exosomes, specifically targeting hair follicle stem cells and skin epidermal cells; Antibody modification: Antibodies targeting specific surface receptors are attached to the surface of exosomes to enhance the specific recognition of exosomes to target cells. Peptide modification: using RGD peptide to enhance the targeting of exosomes to skin cells; Surface charge regulation of exosomes: Changing the charge on the surface of exosomes can regulate their affinity to target cells.

8. The preparation of hair follicle stem cell exosomes according to claim 7, characterized in that: The described load and function enhancement: Gene load: using exosomes as gene carriers to load specific genes and small interfering RNA.

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