CN107828716B - Method for inducing and differentiating sweat gland cells by epidermal stem cells and culture medium group thereof - Google Patents
Method for inducing and differentiating sweat gland cells by epidermal stem cells and culture medium group thereof Download PDFInfo
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- CN107828716B CN107828716B CN201710909244.5A CN201710909244A CN107828716B CN 107828716 B CN107828716 B CN 107828716B CN 201710909244 A CN201710909244 A CN 201710909244A CN 107828716 B CN107828716 B CN 107828716B
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Abstract
The invention discloses a method for inducing and differentiating sweat gland cells by epidermal stem cells, which comprises the following steps: 1) separating epidermal stem cells from in vitro skin tissues, and carrying out subculture on an epidermal stem cell culture medium; 2) subculturing the epidermal stem cells obtained in the step 1), and adding a sweat gland cell induction culture medium for induction differentiation; and 3) carrying out passage and proliferation by using a sweat gland cell culture medium. The invention also provides a corresponding culture medium group. The method effectively avoids ethical disputes, and is helpful for preparing sweat gland cells with uniform cell directional differentiation and proliferation.
Description
Technical Field
The invention relates to the technical field of biological tissue culture, in particular to a method for inducing and differentiating sweat gland cells by epidermal stem cells and a culture medium group thereof.
Background
The skin is the largest organ of the human body and has the functions of protecting the body, removing sweat, feeling cold and heat, pressure and the like. Sweat glands secrete sweat, dissipating body heat. After the wound surface of a large-area burn patient is repaired, sweat glands are lost, so that the temperature regulation function is affected. The artificial skin has only the structure of epidermis and dermis, no sweat gland and other skin accessory. The problem that the tissue engineering skin is difficult to construct sweat glands is to be solved at present. Simulating the mechanism of occurrence of sweat glands to induce the directional differentiation of stem cells into sweat gland cells may be the only way to reconstitute sweat glands. The method has the advantages that the epidermal stem cells are used for inducing and differentiating the sweat gland cells to achieve the purpose of sweat gland cell amplification culture, and the method has a good application prospect, but the cell differentiation and metabolism mechanisms of the epidermal stem cells are not clear, and more uncertain factors need to be overcome for directional differentiation.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a stable and directional differentiation method for inducing differentiated sweat gland cells by epidermal stem cells.
The second object of the present invention is to provide the above-mentioned culture medium set for inducing differentiation of sweat gland cells from epidermal stem cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a method for inducing differentiation of sweat gland cells from epidermal stem cells, comprising the steps of:
1) separating epidermal stem cells from in vitro skin tissues, and carrying out subculture on an epidermal stem cell culture medium;
the epidermal stem cell culture medium is a DMEM culture medium containing hydrocortisone, insulin, adenine, penicillin, human epidermal growth factor, transferrin, glutamic acid, Y-27632 and carboxymethyl chitosan;
2) subculturing the epidermal stem cells obtained in the step 1), removing a culture medium when the fusion degree of the epidermal stem cells reaches 80%, cleaning by using DMEM/F12, and adding a sweat gland cell induction culture medium for induced differentiation;
the sweat gland cells are induced and cultured in a DMEM medium containing human epidermal growth factors, cholera toxin, triiodothyronine and acetylcholine chloride;
3) culturing for several days until polygonal and paving stone-like cells appear in the culture medium, namely sweat gland cells, changing the culture medium into a sweat gland cell culture medium, culturing for several days, and then carrying out passage and proliferation;
the sweat gland cell culture medium is a DMEM culture medium containing EGF, bovine pituitary extract, penicillin and streptomycin.
Further, in the step 1), the in vitro skin tissue is discarded foreskin of healthy children after foreskin cutting.
Further, in step 1), the operation of isolating epidermal stem cells from the excised skin tissue is specifically: taking discarded foreskin of healthy children of 3-6 years old after foreskin cutting, cleaning skin tissue with chloramphenicol under aseptic condition, adding dispase, acting at 4 deg.C for 18-24h, and separating epidermis tissue; cleaning epidermal tissues by sterile PBS, digesting by 0.25% pancreatin-EDTA for 4h, adding an epidermal stem cell culture medium for termination, centrifuging for 5min at 1000r/min, collecting epidermal cells, cleaning for three times by sterile PBS, adding the epidermal stem cell culture medium for heavy suspension, and culturing in a Matrigel-coated culture plate to obtain the epidermal stem cells.
Further, in step 2), the sweat gland cell induction medium is changed every day when differentiation is induced.
Further, the epidermal stem cell culture medium is a DMEM medium containing the following components in final concentration: 0.1-2ng/mL hydrocortisone, 0.01-1ng/mL insulin, 1-5X 10-4mol/L adenine, 50-200IU/mL penicillin, 5-50ng/mL human epidermal growth factor, 2-50 μ g/mL transferrin, 1-10 μ g/mL glutamic acid, 1-20 μ M Y-27632 and 0.01-1mg/mL carboxymethyl chitosan.
Further, the sweat gland cell induction medium is a DMEM medium containing the following components in final concentration: 25-100ng/mL human epidermal growth factor, 0.1-2X 10-10mol/L cholera toxin, 0.5-5X 10-7mol/L triiodothyronine and 2-8X 10-5mol/L acetylcholine chloride.
Further, the sweat gland cell culture medium is a DMEM medium containing the following components in final concentration: 10-100ng/mL EGF, 10-50mg/mL bovine pituitary extract, 50-200U/mL penicillin, and 50-200 μ g/mL streptomycin.
The second purpose of the invention is realized by adopting the following technical scheme:
a culture medium group for inducing differentiated sweat gland cells by epidermal stem cells comprises the following culture media:
epidermal stem cell culture medium: DMEM medium containing hydrocortisone, insulin, adenine, penicillin, human epidermal growth factor, transferrin, glutamic acid, Y-27632 and carboxymethyl chitosan;
sweat gland cell induction culture: DMEM medium containing human epidermal growth factor, cholera toxin, triiodothyronine and acetylcholine chloride;
sweat gland cell culture medium: DMEM medium containing EGF, bovine pituitary extract, penicillin and streptomycin.
Further, the epidermal stem cell culture medium is a DMEM culture medium containing 0.1-2ng/mL hydrocortisone, 0.01-1ng/mL insulin and 1-5 × 10 in terms of final concentration-4mol/L adenine, 50-200IU/mL penicillin, 5-50ng/mL human epidermal growth factor, 2-50 mug/mL transferrin, 1-10 mug/mL glutamic acid, 1-20 mug M Y-27632 and 0.01-1mg/mL carboxymethyl chitosan.
Further, the sweat gland cell induction culture medium is DMEM culture medium containing 25-100ng/mL human epidermal growth factor and 0.1-2 × 10 in terms of final concentration-10mol/L cholera toxin, 0.5-5 × 10-7mol/L triiodothyronine, 2-8 × 10-5mol/L acetylcholine chloride;
the sweat gland cell culture medium is a DMEM culture medium containing the following components in final concentration: 10-100ng/mL EGF, 10-50mg/mL bovine pituitary extract, 50-200U/mL penicillin, and 50-200 μ g/mL streptomycin.
Compared with the prior art, the invention has the beneficial effects that:
1) the invention creatively uses the human in vitro tissue sample as the sampling sample, effectively avoids unnecessary pain of taking blood sample, bone marrow sample and the like, simultaneously avoids ethical concerns, and has long-term significance;
2) according to the method provided by the invention, the epidermal stem cells are conveniently and quickly obtained, the immunity prototype is low, and the used culture medium is serum-free culture, so that the pollution of pathogenic bacteria is avoided, and the safety and reliability are ensured;
3) the invention prepares a culture medium group beneficial to the uniform growth and the consistent differentiation stage of the epidermal stem cells, is suitable for different stages of separation, induced differentiation culture and proliferation culture, and the morphology of the epidermal stem cells has better consistency as seen from an immunofluorescence identification map;
4) the culture medium group provided by the invention can better control the growth, the directional differentiation and the proliferation of cells, and has better application prospect when being used as a matched culture medium group.
Drawings
FIG. 1 is a diagram of immunofluorescence assay of example 1 of the present invention;
FIG. 2 is a diagram showing immunofluorescence assay in example 2 of the present invention;
FIG. 3 is a photograph showing immunofluorescence assay in example 3 of the present invention;
FIG. 4 is a flow cytometer inspection image of examples 1-3 of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
The invention provides a method for inducing and differentiating sweat gland cells by epidermal stem cells, which takes human isolated tissues as raw material sources, and has no ethical disputes in the sampling and preparation processes.
In the following embodiments, reagents and instruments used in the methods not specifically described can be obtained by a commercially available method or a conventional experimental method.
The invention provides a method for inducing and differentiating sweat gland cells by epidermal stem cells, which comprises the following steps:
1) separating epidermal stem cells from in vitro skin tissues, and carrying out subculture on an epidermal stem cell culture medium;
the epidermal stem cell culture medium is a DMEM culture medium containing hydrocortisone, insulin, adenine, penicillin, human epidermal growth factor, transferrin, glutamic acid, Y-27632 and carboxymethyl chitosan;
the epidermal stem cell culture medium does not contain serum, carboxymethyl chitosan is a chitosan derivative, the epidermal stem cell culture medium is used as a carrier, has good compatibility with the epidermal stem cells, and can help the epidermal stem cells to uniformly and morphologically and controllably grow;
2) subculturing the epidermal stem cells obtained in the step 1), removing a culture medium when the fusion degree of the epidermal stem cells reaches 80%, cleaning by using DMEM/F12, and adding a sweat gland cell induction culture medium for induced differentiation;
the sweat gland cells are induced and cultured in a DMEM medium containing human epidermal growth factors, cholera toxin, triiodothyronine and acetylcholine chloride; the sweat gland cell induction culture can uniformly induce the epidermal stem cells to directionally differentiate towards the sweat gland cells;
3) culturing for several days until polygonal and paving stone-like cells appear in the culture medium, namely sweat gland cells, changing the culture medium into a sweat gland cell culture medium, culturing for several days, and then carrying out passage and proliferation;
the sweat gland cell culture medium is a DMEM culture medium containing EGF, bovine pituitary extract, penicillin and streptomycin; the sweat gland cell culture medium is beneficial to uniform and morphologically controllable passage and proliferation of sweat gland cells.
Example 1:
1) preparation of a culture medium:
DMEM medium is taken to prepare a medium containing 0.5ng/mL hydrocortisone, 0.05ng/mL insulin and 1.8 × 10- 4DMEM medium of mol/L adenine, 100IU/mL penicillin, 15ng/mL human epidermal growth factor, 10 mug/mL transferrin, 5 mug/mL glutamic acid, 5 mug M Y-27632 and 0.1mg/mL carboxymethyl chitosan;
sweat gland cell induction culture medium, taking DMEM culture medium, preparing to obtain the product containing 50ng/mL human epidermal growth factor and 1 × 10-10mol/L cholera toxin, 1 × 10-7mol/L Triiodothyronine, 5 × 10-5DMEM medium containing mol/L acetylcholine chloride;
sweat gland cell culture medium: DMEM medium was taken and prepared to contain 50ng/mL EGF, 25mg/mL bovine pituitary extract, 100U/mL penicillin and 100. mu.g/mL streptomycin.
2) Obtaining and culturing of epidermal stem cells
The specific operation of separating the epidermal stem cells from the in vitro skin tissue is as follows: taking discarded foreskin of healthy children of 3-6 years old after foreskin cutting, cleaning skin tissue with chloramphenicol under aseptic condition, adding dispase, acting at 4 deg.C for 18-24h, and separating epidermis tissue; cleaning epidermal tissues by sterile PBS, digesting by 0.25% pancreatin-EDTA for 4h, adding an epidermal stem cell culture medium for termination, centrifuging for 5min at 1000r/min, collecting epidermal cells, cleaning for three times by sterile PBS, adding the epidermal stem cell culture medium for heavy suspension, and culturing in a Matrigel-coated culture plate to obtain epidermal stem cells;
3) induction and culture of sweat gland cells
Subculturing the epidermal stem cells prepared in the step 2), removing the culture medium when the fusion degree of the epidermal stem cells reaches 80%, washing with DMEM/F12 once, and adding sweat gland cells for induction culture to perform induced differentiation;
changing the culture medium every day, culturing for about 7 days until polygonal and paving stone-like cells appear in the culture medium, namely sweat gland cells, changing the culture medium into a sweat gland cell culture medium, culturing for 3 days, and then carrying out passage and proliferation.
Example 2:
example 2 in contrast to example 1,
1) preparation of a culture medium:
the epidermal stem cell culture medium is prepared from DMEM culture medium containing hydrocortisone 0.1ng/mL, insulin 0.01ng/mL and insulin 1 × 10-4DMEM medium of mol/L adenine, 50IU/mL penicillin, 5ng/mL human epidermal growth factor, 2 mug/mL transferrin, 1 mug/mL glutamic acid, 1 mug M Y-27632 and 0.01mg/mL carboxymethyl chitosan;
sweat gland cell induction culture medium, taking DMEM culture medium, preparing to obtain the product containing 25ng/mL human epidermal growth factor and 0.1 × 10-10mol/L cholera toxin, 0.5 × 10-7mol/L Triiodothyronine, 2 × 10-5DMEM medium containing mol/L acetylcholine chloride;
sweat gland cell culture medium: DMEM medium was taken and prepared to contain 10ng/mL EGF, 10mg/mL bovine pituitary extract, 50U/mL penicillin and 50. mu.g/mL streptomycin.
Example 3:
example 3 differs from example 1 in 1) preparation of the medium:
the epidermal stem cell culture medium is prepared from DMEM culture medium containing hydrocortisone (2 ng/mL), insulin (1 ng/mL) and 5 × 10-4DMEM medium of mol/L adenine, 200IU/mL penicillin, 50ng/mL human epidermal growth factor, 50 mug/mL transferrin, 10 mug/mL glutamic acid, 20 mug M Y-27632 and 1mg/mL carboxymethyl chitosan;
sweat gland cell induction culture medium, taking DMEM culture medium, preparing to obtain the culture medium containing 100ng/mL human epidermal growth factor and 2 × 10-10mol/L cholera toxin, 5 × 10-7mol/L Triiodothyronine, 8 × 10-5DMEM medium containing mol/L acetylcholine chloride;
sweat gland cell culture medium: DMEM medium was taken and prepared to contain 100ng/mL EGF, 50mg/mL bovine pituitary extract, 200U/mL penicillin and 200. mu.g/mL streptomycin.
Comparative example 1:
comparative example 1 in contrast to example 1, the sweat gland cell culture medium was KSFM medium containing 10 vt% FBS.
Performance detection and effect evaluation:
detection of cell viability by MTT method
The epidermal stem cells obtained in each example and comparative example were tested by the MTT method, and their activities are shown in the following table:
| example 1 | Example 2 | Example 3 | Comparative example 1 | |
| Activity [% ]] | 96.57 | 83.78 | 80.38 | 97.11 |
Wherein the viability of the cells prepared in example 1 was significantly higher than that of examples 2-3(P <0.05) and was not significantly different from that of comparative example 1 (P >0.05), indicating that the same effect was achieved in serum-free culture as in commercial serum-containing culture.
2. Immunofluorescence identification of sweat gland cell surface markers
The cells obtained in example 1 were digested with 0.25% pancreatin, and the extract was purified to obtain a extract of 1 × 105The inoculation amount of (2) is inoculated in a 12-hole plate, after the plate is adhered to the wall, 4 percent paraformaldehyde is fixed for 2 hours, and PBS is washed for three times;
adding 200 μ L primary anti-dilution solution, sealing at room temperature for 1-2 hr; the primary antibody diluent is PBS containing 10 vt% serum and 0.3 vt% TritonX-100;
discarding primary anti-dilution solution, adding 200 μ L murine CK19 and CK15 primary anti-antibody (dilution 1:100), incubating overnight at 4 deg.C, washing with PBS for 5min for three times and 3 times;
adding 200 μ L FITC labeled anti-mouse secondary antibody (1: 400), reacting at normal temperature in dark for 1h, and washing with PBS; and dyeing the core with PI before machine detection.
As can be seen from the observation result of a fluorescence inverted microscope, the positive markers CEA, CK14, CK18 and CK8 of the sweat gland cells are positively expressed in most sweat gland cells. The fluorescein PE labeled antibody is combined with the CEA, CK14, CK18 and CK8 monoclonal antibodies, and shows red fluorescence in cytoplasm, and the DAPI reagent stains the nucleus of the sweat gland tissue-derived cell to be blue.
Wherein, the detection results of the sweat gland cells prepared in examples 1-3 are shown in FIGS. 1-3. As can be seen from FIGS. 1-3, the CEA, CK14, CK18 and CK8 proteins are specific marker proteins of sweat gland cells, and the CEA, CK14, CK18 and CK8 are positively expressed in extremely high expression levels, which indicates that the induced cells are sweat gland cells. And the positive rate of each protein in example 1 is obviously higher than that in examples 2-3(p < 0.05).
3. Flow cytometry for detecting sweat gland cell surface marker
Digesting epidermal stem cells with 0.25% pancreatin and collecting cells, the number of cells was 2 × 105A plurality of;
resuspending the cell in 1mL PBS, washing for 2 times, 200g, centrifuging for 5min, adding 1mL 70% precooled alcohol into the obtained cell precipitate, and resuspending and fixing for 2 h;
centrifuging, discarding the fixed solution, adding 200 μ L murine CK19, CK15, CK10 primary antibody (dilution 1:100), and incubating at room temperature for 30 min;
washing with 1mL of PBS, and respectively incubating with FITC labeled secondary antibodies (dilution is 1:200) for 1h at normal temperature in a dark place;
after washing twice with PBS, discarding PBS, adding a proper amount of PBS to resuspend cells according to the cell amount, and detecting by using a flow cytometer.
Wherein, the detection results of the sweat gland cells obtained in examples 1-3 are shown in FIG. 4.
Flow cytometry analysis shows that the positive expression amount of CK14 protein in sweat gland cells of example 1 is more than 95%, the positive expression amount of CEA protein is more than 75%, and the negative expression amount of MSX-1 protein is less than 2%; example 2 in sweat gland cells CK14 protein positive expression of more than 82%, CEA protein positive expression of more than 65%, and MSX-1 protein negative expression of less than 4%; example 3 the positive expression level of CK14 protein in sweat gland cell is more than 80%, the positive expression level of CEA protein is more than 62%, and the negative expression level of MSX-1 protein is less than 5%, all of which accord with the expression result of sweat gland cell surface marker. And the expression of each protein in example 1 is obviously better than that in examples 2-3(p < 0.05).
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (8)
1. A method for inducing differentiation of sweat gland cells from epidermal stem cells, comprising the steps of:
1) separating epidermal stem cells from in vitro skin tissues, and carrying out subculture on an epidermal stem cell culture medium;
the epidermal stem cell culture medium is a DMEM culture medium containing hydrocortisone, insulin, adenine, penicillin, human epidermal growth factor, transferrin, glutamic acid, Y-27632 and carboxymethyl chitosan;
2) subculturing the epidermal stem cells obtained in the step 1), removing a culture medium when the fusion degree of the epidermal stem cells reaches 80%, cleaning by using DMEM/F12, and adding a sweat gland cell induction culture medium for induced differentiation;
the sweat gland cell induction culture medium is DMEM culture medium containing 25-100ng/mL human epidermal growth factor and 0.1-2 × 10-10mol/L cholera toxin, 0.5-5 × 10-7mol/L triiodothyronine, 2-8 × 10-5mol/L acetylcholine chloride;
3) culturing for several days until polygonal and paving stone-like cells appear in the culture medium, namely sweat gland cells, changing the culture medium into a sweat gland cell culture medium, culturing for several days, and then carrying out passage and proliferation;
the sweat gland cell culture medium is a DMEM culture medium containing the following components in final concentration: 10-100ng/mLEGF, 10-50mg/mL bovine pituitary extract, 50-200U/mL penicillin and 50-200 μ g/mL streptomycin.
2. The method of claim 1, wherein in step 1), the excised skin tissue is discarded after prepuce cutting in healthy young children.
3. The method of claim 1, wherein the isolating epidermal stem cells from the excised skin tissue in step 1) is performed by: taking discarded foreskin of healthy children of 3-6 years old after foreskin cutting, cleaning skin tissue with chloramphenicol under aseptic condition, adding dispase, acting at 4 deg.C for 18-24h, and separating epidermis tissue; washing epidermal tissues by sterile PBS, digesting by 0.25% pancreatin-EDTA for 4h, adding an epidermal stem cell culture medium to terminate, centrifuging for 5min at the speed of 1000r/min, collecting epidermal cells, washing for three times by sterile PBS, adding the epidermal stem cell culture medium to resuspend, and culturing in a Matrigel-coated culture plate to obtain the epidermal stem cells.
4. The method of claim 1, wherein in the step 2), the sweat gland cell induction medium is changed every day when inducing differentiation.
5. The method according to any one of claims 1 to 4, wherein the epidermal stem cell culture medium is DMEM medium containing hydrocortisone at 0.1-2ng/mL, insulin at 0.01-1ng/mL, and insulin at 1-5 × 10 at final concentration-4mol/L adenine, 50-200IU/mL penicillin, 5-50ng/mL human epidermal growth factor, 2-50 mug/mL transferrin, 1-10 mug/mL glutamic acid, 1-20 mug M Y-27632 and 0.01-1mg/mL carboxymethyl chitosan.
6. A culture medium group for inducing differentiated sweat gland cells by epidermal stem cells comprises the following culture media:
epidermal stem cell culture medium: DMEM medium containing hydrocortisone, insulin, adenine, penicillin, human epidermal growth factor, transferrin, glutamic acid, Y-27632 and carboxymethyl chitosan;
sweat gland cell induction culture: DMEM medium containing human epidermal growth factor, cholera toxin, triiodothyronine and acetylcholine chloride;
sweat gland cell culture medium: DMEM medium containing EGF, bovine pituitary extract, penicillin and streptomycin.
7. The medium set according to claim 6,
the epidermal stem cell culture medium is a DMEM culture medium containing hydrocortisone 0.1-2ng/mL, insulin 0.01-1ng/mL and insulin 1-5 × 10 in final concentration-4mol/L adenine, 50-200IU/mL penicillin, 5-50ng/mL human epidermal growth factor, 2-50 mug/mL transferrin, 1-10 mug/mL glutamic acid, 1-20 mug M Y-27632 and 0.01-1mg/mL carboxymethyl chitosan.
8. The medium set according to claim 6,
the sweat gland cell induction culture medium is DMEM culture medium containing 25-100ng/mL human epidermal growth factor and 0.1-2 × 10-10mol/L cholera toxin, 0.5-5 × 10-7mol/L triiodothyronine, 2-8 × 10-5mol/L acetylcholine chloride;
the sweat gland cell culture medium is a DMEM culture medium containing the following components in final concentration: 10-100ng/mLEGF, 10-50mg/mL bovine pituitary extract, 50-200U/mL penicillin and 50-200 μ g/mL streptomycin.
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