CN112342186A - Culture method of hair follicle stem cells - Google Patents
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Abstract
The invention discloses a hair follicle stem cell culture method, which is obtained by culturing, separating and purifying hair follicle stem cell culture medium with an outer hair follicle root sheath bulge part of a complete hair follicle. The culture method has the advantages of short period, simple operation and high feasibility, and is suitable for commercialization.
Description
Technical Field
The invention relates to the fields of genetic engineering technology and biological medicines, in particular to a hair follicle stem cell culture method.
Background
Hair Follicle Stem Cells (HFSCs) are a group of pluripotent stem cells that are present in the outer root sheath bulge of hair follicles and are capable of self-renewal and proliferation, and are capable of differentiating into hair follicles, sebaceous glands and epidermis, playing an important role in the regeneration of hairs. Studies have shown that human hair follicle stem cells express a variety of cell surface molecular markers such as CD200, keratin 15(keratin 15, K15), keratin 19(keratin 19, K19), integrin α 6, integrin β 1, Nestin, etc., but do not express the endothelial cell marker molecule CD 31; in addition, the marker molecule CD34 of the mouse hair follicle stem cell is not expressed. By using the positive and negative marker molecules, researchers can better separate and purify hair follicle stem cells, which accelerates the research process of the hair follicle stem cells. Compared with embryonic stem cells and other adult stem cells, the hair follicle stem cells have the advantages of rich sources, convenient material taking, no damage to organisms, no ethical problem and the like. The literature reports that the transplanted healthy hair follicle stem cells have the potential of repairing damaged hair follicles by transplanting hair follicle stem cells to make nude mice grow hairs.
However, hair follicle stem cells may have a complex set of mechanisms in promoting hair regrowth. Garza et al found that there was no significant difference in the number of hair follicle stem cells in the patient's hair-lost tissue and scalp tissue that did not develop hair loss, but the hair follicle stem cells in the hair-lost scalp tissue failed to produce source cells for hair growth, indicating that the hair follicle stem cells in the hair-lost scalp tissue may be defective. Matsumura et al, showed that accumulated DNA damage from hair follicle stem cells prevented their function. Further studies have shown that the proliferative differentiation of hair follicle stem cells is influenced by their surrounding microenvironment; signals from surrounding hair follicles affect the activity of hair follicle stem cells, thereby stimulating hair follicle growth. Therefore, research on the action mechanism of the hair follicle stem cells for promoting the hair follicle regeneration is helpful for promoting the clinical application of the hair follicle stem cells. In addition, although the hair follicle stem cells have definite cell surface marker molecules, the operation of separating and culturing the hair follicle stem cells is complex and the time period is long. In view of the above, the present invention studies the culture method of hair follicle stem cells and explores the feasibility of using allogeneic hair follicle stem cells to treat pathological alopecia.
Disclosure of Invention
The invention aims to research a culture method of hair follicle stem cells and explore the feasibility of treating pathological alopecia by utilizing allogeneic hair follicle stem cells.
A method for culturing the stem cells of hair follicle features that the external root sheath of hair follicle is cultured in the culture medium of stem cells of hair follicle, which is then separated and purified.
The culture method is characterized by comprising the following steps:
(1) the intact hair follicle is taken from the occipital part of the back of the brain and is repeatedly washed by PBS (phosphate buffer solution) of penicillin-streptomycin;
(2) cutting off the hair follicle outer root sheath bulge part by adopting a microscopical scissors under a dissecting microscope, and putting the hair follicle outer root sheath bulge part into a culture dish; adding hair follicle stem cell culture medium, at 37 deg.C and 5% CO2Culturing an incubator;
(3) supplementing 1.5mL of equivalent culture medium the next day, observing whether the hair follicle external root sheath bulge has fusiform growing cells to climb out under an inverted microscope, and carrying out subculture after the cells are fused to 80%;
(4) digesting the hair follicle stem cells, and preparing into 1.0 × 10 hair follicle stem cell culture solution5And (4) counting the number of the hair follicle stem cells in each mL.
(5) 1.2% agarose and DMEM/F12 medium were mixed at a ratio of 1:1, and 2ml of the mixture was poured into a 35cm2 petri dish and allowed to cool and solidify, and then placed in an incubator for use.
(6) 0.7% agarose and hair follicle stem cell culture medium are mixed in a sterile test tube according to the proportion of 1:1, 1/10 volumes of hair follicle stem cell suspension are added into the tube, the mixture is fully mixed and injected into a culture dish of a culture medium I, and the adding amount is equal to that of the culture medium I.
(7) The culture medium is changed once every 2 days, the culture is carried out for 10 days in total,
further, the hair follicle stem cell culture solution contains 10% FBS, 100U/mL streptomycin double antibody and 2ng/mlbFGF DMEM/F12.
Further, after 1.2% agarose and DMEM/F12 medium were mixed at a ratio of 1:1, 2ml of the mixture was poured into a 35cm2 petri dish and, after cooling and solidification, placed in an incubator for use.
Further, the digestive juice digested by the hair follicle stem cells contains 0.1% of pancreatin and 0.008% of EDTA phosphate buffer solution in percentage by mass.
Further, the digestion conditions were 37 ℃ for 3-5 minutes.
Further, the method also comprises the expression of surface marker molecules of the hair follicle stem cells, and the expression of the surface marker molecules and the negative marker molecules of the hair follicle stem cells is identified by adopting flow cytometry: digesting and collecting the hair follicle stem cells of the 3 rd generation by 0.25% of trypsin, washing the hair follicle stem cells by PBS for 1 time, centrifuging and collecting the cells, resuspending the cells by a PBS solution containing 1% BSA, respectively adding K15, K19, CD200, integrin beta 1, CD31 and CD34 fluorescent antibodies, incubating the cells for 30min at room temperature in a dark place, and detecting the expression condition of the marker molecules by a flow cytometer after washing the cells by PBS.
Further, the identification of immunogenicity of hair follicle stem cells includes: 1) WB assay hair follicle stem cell MHC-class II molecule expression: collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract total protein and quantifying; adding 15 mu g of protein sample into 2 xSDS gel loading buffer solution, boiling for 5min, centrifuging at 6000rpm for 3min, taking supernatant for loading, transferring the protein on the gel to a PVDF membrane after electrophoresis, sealing at room temperature for 2h after membrane conversion, putting the PVDF membrane into a hybridization bag, respectively adding a primary antibody (HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH) which is diluted properly at 4 ℃ overnight, adding a secondary antibody labeled by HRP corresponding to a primary antibody species, oscillating at room temperature for incubation for 1h, adding a developing solution for developing color after the filter membrane is rinsed, and putting the filter membrane into a gel imager for exposure and imaging; analyzing the optical density value of the target zone by using a Quantity One software processing system;
2) detecting the expression of the hair follicle stem cell MHC-II molecules by real-time fluorescent quantitative PCR: collecting hair follicle stem cells, extracting total mRNA of the cells by a Trizol method, and identifying and quantifying by an ultraviolet spectrophotometer; the total RNA was reverse transcribed into cDNA using a reverse transcription reaction kit.
The invention has the beneficial effects that:
the culture method is simple and convenient to operate and short in time period, and the hair follicle stem cells cultured by the method are used for carrying out allogenic normal human hair follicle stem cell combined drug molecules to build a new thought suitable for a hair follicle repairing microenvironment.
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FIG. 1 shows the skin HE of each group of nude mice after 5w injection;
Detailed Description
The following detailed description further describes the present invention for the purpose of illustrating the technical solutions and objects of the present invention.
A method for culturing the stem cells of hair follicle features that the external root sheath of hair follicle is cultured in the culture medium of stem cells of hair follicle, which is then separated and purified.
The culture method according to claim 1, comprising the steps of:
(1) the intact hair follicle is taken from the occipital part of the back of the brain and is repeatedly washed by PBS (phosphate buffer solution) of penicillin-streptomycin;
(2) cutting off the hair follicle outer root sheath bulge part by adopting a microscopical scissors under a dissecting microscope, and putting the hair follicle outer root sheath bulge part into a culture dish; adding hair follicle stem cell culture medium, at 37 deg.C and 5% CO2Culturing an incubator;
(3) supplementing 1.5mL of equivalent culture medium the next day, observing whether the hair follicle external root sheath bulge has fusiform growing cells to climb out under an inverted microscope, and carrying out subculture after the cells are fused to 80%;
(4) the above hair follicle stem cells were digested, prepared into a 1.0X 105 hair follicle stem cell suspension per mL using a hair follicle stem cell culture medium, and counted.
(5) 1.2% agarose and DMEM/F12 medium were mixed at a ratio of 1:1, and 2ml of the mixture was poured into a 35cm2 petri dish and allowed to cool and solidify, and then placed in an incubator for use.
(6) 0.7% agarose and hair follicle stem cell culture medium are mixed in a sterile test tube according to the proportion of 1:1, 1/10 volumes of hair follicle stem cell suspension are added into the tube, the mixture is fully mixed and injected into a culture dish of a culture medium I, and the adding amount is equal to that of the culture medium I.
(7) The culture medium is changed once every 2 days, the culture is carried out for 10 days in total,
further, the hair follicle stem cell culture solution contains 10% FBS, 100U/mL streptomycin double antibody and 2ng/mL bFGF in DMEM/F12.
Further, the medium I was prepared by mixing 1.2% agarose and DMEM/F12 medium at a ratio of 1:1, and then injecting 2ml of the mixture into 35cm2And (4) placing the culture dish into an incubator for later use after cooling and solidification.
Further, the digestive juice digested by the hair follicle stem cells contains 0.1% of pancreatin and 0.008% of EDTA phosphate buffer solution in percentage by mass.
Further, the digestion conditions were 37 ℃ for 3-5 minutes.
Further, the method also comprises the expression of surface marker molecules of the hair follicle stem cells, and the expression of the surface marker molecules and the negative marker molecules of the hair follicle stem cells is identified by adopting flow cytometry: digesting and collecting the hair follicle stem cells of the 3 rd generation by 0.25% of trypsin, washing the hair follicle stem cells by PBS for 1 time, centrifuging and collecting the cells, resuspending the cells by a PBS solution containing 1% BSA, respectively adding K15, K19, CD200, integrin beta 1, CD31 and CD34 fluorescent antibodies, incubating the cells for 30min at room temperature in a dark place, and detecting the expression condition of the marker molecules by a flow cytometer after washing the cells by PBS.
Further, the identification of immunogenicity of hair follicle stem cells includes: 1) WB assay hair follicle stem cell MHC-class II molecule expression: collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract total protein and quantifying; adding 15 mu g of protein sample into 2 xSDS gel loading buffer solution, boiling for 5min, centrifuging at 6000rpm for 3min, taking supernatant for loading, transferring the protein on the gel to a PVDF membrane after electrophoresis, sealing at room temperature for 2h after membrane conversion, putting the PVDF membrane into a hybridization bag, respectively adding a primary antibody (HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH) which is diluted properly at 4 ℃ overnight, adding a secondary antibody labeled by HRP corresponding to a primary antibody species, oscillating at room temperature for incubation for 1h, adding a developing solution for developing color after the filter membrane is rinsed, and putting the filter membrane into a gel imager for exposure and imaging; analyzing the optical density value of the target zone by using a Quantity One software processing system;
2) detecting the expression of the hair follicle stem cell MHC-II molecules by real-time fluorescent quantitative PCR: collecting hair follicle stem cells, extracting total mRNA of the cells by a Trizol method, and identifying and quantifying by an ultraviolet spectrophotometer; the total RNA was reverse transcribed into cDNA using a reverse transcription reaction kit.
Isolated culture of hair follicle stem cells
1) Separating and culturing human hair follicle stem cells: 10 healthy volunteers were recruited, 8-10 intact hair follicles were extracted from the occipital region of the back of the brain of each volunteer, and washed repeatedly 3 times with a PBS solution containing high-concentration penicillin and streptomycin. Cutting off the bulge of the external root sheath of the hair follicle by adopting a microsurgical scissors under a dissecting microscope, and putting the bulge into a 35cm2 culture dish; adding 1.5ml of hair follicle stem cell culture medium(DMEM/F12 medium containing 10% FBS, 100U/mL streptomycin diabody, 2ng/mL bFGF), 5% CO at 37 ℃2And (5) incubator culture. Adding 1.5mL of culture medium on the next day, observing whether the hair follicle external root sheath bulge has fusiform growing cells to climb out under an inverted microscope, and carrying out passage after the cells are fused to 80%. Cells subcultured to passage 3 were used for subsequent experiments.
2) Identifying the expression of the hair follicle stem cell surface marker molecule and the negative marker molecule by flow cytometry: 0.25% trypsin digestion collected passage 3 hair follicle stem cells, PBS washing 1 times, centrifugal cell collection, containing 1% BSA PBS solution heavy suspension. Respectively adding K15, K19, CD200, integrin beta 1, CD31 and CD34 fluorescent antibodies, incubating the labeled cells for 30min at room temperature in a dark place, washing the labeled cells with PBS, and detecting the expression condition of the labeled molecules by a flow cytometer.
2. Immunogenicity identification of hair follicle stem cells
1) WB assay hair follicle stem cell MHC-class II molecule expression: collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract and quantify total protein. Adding 15 mu g of protein sample into 2 xSDS gel loading buffer solution, boiling for 5min, centrifuging at 6000rpm for 3min, taking supernatant for loading, transferring the protein on the gel to a PVDF membrane after electrophoresis, sealing at room temperature for 2h after membrane conversion, putting the PVDF membrane into a hybridization bag, respectively adding a primary antibody (HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH) which is diluted properly at 4 ℃ overnight, adding a secondary antibody labeled by HRP corresponding to the primary antibody species, oscillating at room temperature for incubation for 1h, adding a developing solution for developing color after the filter membrane is rinsed, and putting into a gel imager for exposure and imaging. And analyzing the optical density value of the target zone by using a Quantity One software processing system.
2) Detecting the expression of the hair follicle stem cell MHC-II molecules by real-time fluorescent quantitative PCR: collecting hair follicle stem cells, extracting cell total mRNA by a Trizol method, and identifying and quantifying by an ultraviolet spectrophotometer. Using a reverse transcription reaction kit (
RT reagent Kit with gDNA Eraser, TAKARA) reverse transcribes total RNA to cDNA. HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference were designed using Primer5.0 software based on Genebank sequencesGAPDH primers were synthesized by the same company. The qRT-PCR reaction was performed according to the kit (SYBR Premix ExTaqTM II, TAKARA) instructions. HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH primers are shown in the following table:
TABLE 1 HLA-DPA1, HLA-DQA1, HLA-DRA1 and internal reference GAPDH primer sequences
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (9)
1. A method for culturing the stem cells of hair follicle features that the external root sheath of hair follicle is cultured in the culture medium of stem cells of hair follicle, which is then separated and purified.
2. The culture method according to claim 1, comprising the steps of:
(1) the intact hair follicle is taken from the occipital part of the back of the brain and is repeatedly washed by PBS (phosphate buffer solution) of penicillin-streptomycin;
(2) cutting off the hair follicle outer root sheath bulge part by adopting a microscopical scissors under a dissecting microscope, and putting the hair follicle outer root sheath bulge part into a culture dish; adding hair follicle stem cell culture medium, at 37 deg.C and 5% CO2Culturing an incubator;
(3) supplementing 1.5mL of equivalent culture medium the next day, observing whether the hair follicle external root sheath bulge has fusiform growing cells to climb out under an inverted microscope, and carrying out subculture after the cells are fused to 80%;
(4) digesting the hair follicle stem cells, preparing 1.0 multiplied by 105 per mL hair follicle stem cell suspension by using a hair follicle stem cell culture solution, and counting;
(5) 1.2% agarose and DMEM/F12 medium were mixed at a ratio of 1:1, and 2ml of the mixture was poured into 35cm2Placing the culture dish into an incubator for later use after cooling and solidification;
(6) mixing 0.7% agarose and a hair follicle stem cell culture medium in a ratio of 1:1 in a sterile test tube, adding 1/10 volumes of hair follicle stem cell suspension into the tube, fully and uniformly mixing, injecting into a culture dish of a culture medium I, and adding the culture medium I in an amount equal to that of the culture medium I;
(7) the medium was changed once every 2 days for a total of 10 days.
3. The method according to claim 2, wherein the hair follicle stem cell culture solution contains 10% FBS, 100U/mL streptomycin diabody, 2ng/mlbFGF in DMEM/F12.
4. The culture method according to claim 2, wherein the medium I is prepared by mixing 1.2% agarose and DMEM/F12 medium at a ratio of 1:1, and then injecting 2ml of the mixture into 35cm2And (4) placing the culture dish into an incubator for later use after cooling and solidification.
5. The culture method according to claim 2, wherein the digestive fluid obtained by digesting the hair follicle stem cells contains 0.1% by mass of pancreatin and 0.008% by mass of EDTA in phosphate buffer.
6. The culture method according to claim 5, wherein the digestion is carried out at 37 ℃ for 3 to 5 minutes.
7. The culture method according to any one of claims 1 to 6, further comprising expressing surface marker molecules of the hair follicle stem cells, and identifying the expression of the surface marker molecules and negative marker molecules of the hair follicle stem cells by flow cytometry: digesting and collecting the hair follicle stem cells of the 3 rd generation by 0.25% of trypsin, washing the hair follicle stem cells by PBS for 1 time, centrifuging and collecting the cells, resuspending the cells by a PBS solution containing 1% BSA, respectively adding K15, K19, CD200, integrin beta 1, CD31 and CD34 fluorescent antibodies, incubating the cells for 30min at room temperature in a dark place, and detecting the expression condition of the marker molecules by a flow cytometer after washing the cells by PBS.
8. The method of any one of claims 1 to 6, wherein the identification of the immunogenicity of the hair follicle stem cells comprises: 1) WB assay hair follicle stem cell MHC-class II molecule expression: collecting hair follicle stem cells, adding a cell total protein extraction reagent to extract total protein and quantifying; adding a 15 mu g protein sample into 2 xSDS gel sample adding buffer solution, boiling for 5min, centrifuging for 3min at 6000rpm, taking supernatant for sample loading, transferring the protein on the gel to a PVDF membrane after electrophoresis, sealing for 2h at room temperature after membrane conversion, putting the PVDF membrane into a hybridization bag, respectively adding a primary antibody with proper dilution at 4 ℃ overnight, adding a secondary antibody marked by HRP corresponding to the species of the primary antibody, oscillating and incubating for 1h at room temperature, rinsing the filter membrane, adding a developing solution for color development, and putting the filter membrane into a gel imager for exposure and imaging; analyzing the optical density value of the target zone by using a Quantity One software processing system;
2) detecting the expression of the hair follicle stem cell MHC-II molecules by real-time fluorescent quantitative PCR: collecting hair follicle stem cells, extracting total mRNA of the cells by a Trizol method, and identifying and quantifying by an ultraviolet spectrophotometer; the total RNA was reverse transcribed into cDNA using a reverse transcription reaction kit.
9. The culture method according to claim 8, wherein the primary antibody is HLA-DPA1, HLA-DQA1, HLA-DRA1, and internal reference GAPDH.
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| CN113564110A (en) * | 2021-08-19 | 2021-10-29 | 上海纳米技术及应用国家工程研究中心有限公司 | Primary separation method of hair follicle stem cells |
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Inventor after: Zhou Xiang Inventor after: Li Shenghua Inventor before: Diao Boqiong Inventor before: Zhou Xiang Inventor before: Li Shenghua |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |