CN116814555A - Genetically modified hair follicle stem cells, culture method and application - Google Patents
Genetically modified hair follicle stem cells, culture method and application Download PDFInfo
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- CN116814555A CN116814555A CN202310814262.0A CN202310814262A CN116814555A CN 116814555 A CN116814555 A CN 116814555A CN 202310814262 A CN202310814262 A CN 202310814262A CN 116814555 A CN116814555 A CN 116814555A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0666—Mesenchymal stem cells from hair follicles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2510/00—Genetically modified cells
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Abstract
The invention belongs to the technical field of cell culture, and discloses a genetically modified hair follicle stem cell, a culture method and application. The culture method comprises the following steps: obtaining hair follicle stem cells and culturing; introducing a desired gene into the cultured hair follicle stem cells, and transducing or transfecting the hair follicle stem cells with the desired gene involved in melanin synthesis by a selected gene delivery method; selecting successfully transduced or transfected hair follicle stem cells by a marker gene or reporter gene; amplifying and culturing the successfully transduced or transfected hair follicle stem cells to obtain genetically modified hair follicle stem cells; the melanogenesis ability of genetically modified hair follicle stem cells was assessed. The invention promotes the generation of melanin by modifying hair follicle stem cells, can be used for stimulating hair to naturally blacken, and avoids using artificial hair dye which can damage hair structure or scalp.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a genetically modified hair follicle stem cell, a culture method and application.
Background
Hair Follicle Stem Cells (HFSCs) are primitive cells in hair follicles that are located in the perifollicular aspect of a human hair follicle, with the outermost layer near the point of attachment of the pili muscles. Research shows that the hair follicle stem cells not only can differentiate to form hair follicles, but also can effectively treat alopecia; but also participate in the process of forming the epidermal tissue. The research of hair follicle stem cells has important theoretical and clinical significance on the pathogenesis of alopecia, hair follicle tissue engineering and stem cell or gene therapy of alopecia. The hair follicle stem cells have the characteristics of undifferentiated property, self-renewal capacity, strong in vitro proliferation capacity and the like. The hair follicle stem cells in vitro culture research show high clonogenic capacity and have high regeneration potential. Since hair follicle stem cells are derived from hair, the hair follicle stem cells can be directly obtained from a patient, are extremely abundant in number, have no complications and are completely noninvasive to the patient. How to promote melanin production by modifying hair follicle stem cells to solve the problems of blackening and whitening of hair is the focus of current research.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a genetically modified hair follicle stem cell, a culture method and application.
The technical scheme adopted by the invention is as follows:
a method of culturing genetically modified hair follicle stem cells, comprising the steps of: s1, obtaining hair follicle stem cells and culturing; s2, introducing a required gene involved in melanin synthesis into the cultured hair follicle stem cells, and transduction or transfection of the required gene involved in melanin synthesis into the hair follicle stem cells by a selected gene transfer method to enable the required gene to be inserted or expressed in the cells; s3, selecting successfully transduced or transfected hair follicle stem cells through a marker gene or a reporter gene; amplifying and culturing the successfully transduced or transfected hair follicle stem cells to obtain genetically modified hair follicle stem cells; s4, evaluating melanin generation capacity of the genetically modified hair follicle stem cells.
Preferably, the step S1 specifically includes the following steps: s11, taking out the hair follicle from the scalp tissue; s12, dissecting hair follicles and treating the dissected hair follicles with an enzyme solution to obtain hair follicle stem cells; s13, enriching hair follicle stem cells through cell sorting; s14, culturing hair follicle stem cells in a culture medium; s15, periodically replacing a culture medium and subculturing to enlarge a cell population; s16, identifying the separated cells and confirming the identity of the hair follicle stem cells.
Preferably, the enzyme solution in the step S12 is trypsin, collagenase or proteinase.
Preferably, the enrichment of hair follicle stem cells is performed in step S13 by fluorescence activated cell sorting or magnetic activated cell sorting.
Preferably, the step S16 identifies hair follicle stem cells by a specific marker, which is CD200, CD117 (c-Kit), nestin or CD34.
Preferably, the culture medium includes growth factors and supplements that support the maintenance and expansion of hair follicle stem cells, and is placed on a culture dish or plate coated with extracellular matrix proteins.
Preferably, the hair follicle stem cells are cultured in a humidified incubator at 37 ℃ plus 5% carbon dioxide.
Preferably, the desired gene for melanin synthesis in step S2 is MITF or TYR.
Preferably, the step S4 evaluates the melanogenesis ability of the genetically modified hair follicle stem cells by spectrophotometrically measuring melanin content or visually evaluating pigmentation.
A genetically modified hair follicle stem cell is obtained by the culture method.
The application of the genetically modified hair follicle stem cells is applied to the white-to-black hair.
The beneficial effects of the invention are as follows:
according to the culture method of the genetically modified hair follicle stem cells, provided by the invention, genes (MITF or TYR) involved in melanin synthesis are introduced into the hair follicle stem cells, and the generation of melanin is promoted by modifying the hair follicle stem cells, so that the hair follicle stem cells can be used for stimulating hair to naturally blacken, and artificial hair dyes which possibly damage hair structures or scalp are avoided. The obtained hair follicle stem cells come from cells of the patient, so that personalized treatment is possible, and the risk of immune response is reduced to the greatest extent.
Detailed Description
The invention will be further illustrated with reference to specific examples. It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
The embodiment provides a culture method of genetically modified hair follicle stem cells, which comprises the following steps:
s1, obtaining hair follicle stem cells and culturing the hair follicle stem cells.
The step S1 specifically comprises the following steps:
s11, taking out the hair follicle from the scalp tissue; hair follicle stem cells are contained in hair follicles, and are used as targets for genetic modification;
s12, carefully dissecting hair follicles from surrounding tissues with fine scissors or forceps under a stereo microscope, and then washing in a suitable buffer or medium to remove any debris or blood. The dissected hair follicle is treated with an enzyme solution, allowing the tissue to separate and release individual cells. Enzymes commonly used for treatment include trypsin, collagenase or protease;
s13, enrichment of hair follicle stem cells is carried out through fluorescence activated cell sorting or magnetic activated cell sorting;
s14, culturing hair follicle stem cells in a culture medium; the culture medium comprises growth factors and supplements for supporting the maintenance and expansion of hair follicle stem cells, such as Fetal Bovine Serum (FBS), epidermal Growth Factor (EGF) or basic fibroblast growth factor (bFGF); the medium is placed on a dish or plate coated with extracellular matrix proteins.
S15, periodically replacing a culture medium and subculturing to enlarge a cell population;
s16, identifying the separated cells and confirming the identity of the hair follicle stem cells. Specifically, hair follicle stem cells can be identified by specific markers, either CD200, CD117 (c-Kit), nestin, or CD34, or by gene expression analysis.
The above culture process of hair follicle stem cells is carried out in a humidified incubator with 5% carbon dioxide at 37 ℃. Cell growth and viability need to be monitored during culture.
S2, introducing a desired gene involved in melanin synthesis (e.g., MITF or TYR) into cultured hair follicle stem cells, gene delivery is accomplished by a variety of methods, including viral vectors (e.g., lentiviruses or retroviruses) or non-viral methods (e.g., electroporation or lipid-based transfection). According to the selected gene delivery method, a desired gene involved in melanin synthesis is transduced or transfected into hair follicle stem cells so that the desired gene can be inserted or expressed in the cells.
S3, selecting successfully transduced or transfected hair follicle stem cells through a marker gene or a reporter gene; the successfully transduced or transfected hair follicle stem cells are subjected to expansion culture to obtain genetically modified hair follicle stem cells to obtain a sufficient number of genetically modified hair follicle stem cells for further experimentation.
S4, evaluating melanin generation capacity of the genetically modified hair follicle stem cells. Specifically, the melanogenesis ability of genetically modified hair follicle stem cells is assessed by spectrophotometrically measuring melanin content or visually assessing pigmentation.
The embodiment also provides a genetically modified hair follicle stem cell, which is obtained by adopting the culture method.
The embodiment also provides an application of the genetically modified hair follicle stem cells in turning white hair into black hair of a person.
The invention is not limited to the alternative embodiments described above, but any person may derive other various forms of products in the light of the present invention. The above detailed description should not be construed as limiting the scope of the invention, which is defined in the claims and the description may be used to interpret the claims.
Claims (10)
1. A method of culturing genetically modified hair follicle stem cells, comprising the steps of:
s1, obtaining hair follicle stem cells and culturing;
s2, introducing a required gene involved in melanin synthesis into the cultured hair follicle stem cells, and transduction or transfection of the required gene involved in melanin synthesis into the hair follicle stem cells by a selected gene transfer method to enable the required gene to be inserted or expressed in the cells;
s3, selecting successfully transduced or transfected hair follicle stem cells through a marker gene or a reporter gene; amplifying and culturing the successfully transduced or transfected hair follicle stem cells to obtain genetically modified hair follicle stem cells;
s4, evaluating melanin generation capacity of the genetically modified hair follicle stem cells.
2. The method of culturing genetically modified hair follicle stem cells of claim 1, wherein: the step S1 specifically comprises the following steps:
s11, taking out the hair follicle from the scalp tissue;
s12, dissecting hair follicles and treating the dissected hair follicles with an enzyme solution to obtain hair follicle stem cells;
s13, enriching hair follicle stem cells through cell sorting;
s14, culturing hair follicle stem cells in a culture medium;
s15, periodically replacing a culture medium and subculturing to enlarge a cell population;
s16, identifying the separated cells and confirming the identity of the hair follicle stem cells.
3. The method of culturing genetically modified hair follicle stem cells of claim 2, wherein: the enzyme solution in the step S12 is trypsin, collagenase or proteinase.
4. The method of culturing genetically modified hair follicle stem cells of claim 2, wherein: the enrichment of hair follicle stem cells is performed by fluorescence activated cell sorting or magnetic activated cell sorting in step S13.
5. The method of culturing genetically modified hair follicle stem cells of claim 2, wherein: the hair follicle stem cells are identified in step S16 by a specific marker, which is CD200, CD117 (c-Kit), nestin or CD34.
6. The method of culturing genetically modified hair follicle stem cells of claim 2, wherein: the culture medium includes growth factors and supplements that support the maintenance and expansion of hair follicle stem cells, and is placed on a culture dish or plate coated with extracellular matrix proteins.
7. The method of culturing genetically modified hair follicle stem cells of claim 1, wherein: the desired gene for melanin synthesis in step S2 is MITF or TYR.
8. The method of culturing genetically modified hair follicle stem cells of claim 1, wherein: the melanin production capacity of the genetically modified hair follicle stem cells is assessed in step S4 by spectrophotometrically measuring the melanin content or visually assessing pigmentation.
9. A genetically modified hair follicle stem cell, characterized in that: obtained by the culture method according to any one of claims 1 to 8.
10. The use of genetically modified hair follicle stem cells as described in claim 9, wherein: is used for turning white hair into black hair.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117646052A (en) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | Extraction and preparation method of stem cell active factor preparation for turning white hair into black hair |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117646052A (en) * | 2023-12-04 | 2024-03-05 | 安徽科门生物科技有限公司 | Extraction and preparation method of stem cell active factor preparation for turning white hair into black hair |
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