CN112725259A - Induction medium and induction method for differentiation of stem cells into sweat gland-like cells - Google Patents
Induction medium and induction method for differentiation of stem cells into sweat gland-like cells Download PDFInfo
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Abstract
The invention discloses an induction culture medium for differentiation of stem cells into sweat gland-like cells, belonging to the technical field of stem cells. The induction medium is DMEM/F-12 cell culture medium containing the following components: fetal bovine serum, epidermal growth factor, basic fibroblast growth factor, recombinant human ectodermal dysplasia A1 protein, recombinant human Wnt3a protein, bone morphogenetic protein 4, hepatocyte growth factor, triiodothyronine, hemisuccinylhydrocortisone, insulin-transferrin-sodium selenite, L-glutamine and penicillin/streptomycin double antibiotic. The sweat gland induction culture medium can effectively induce stem cells, particularly mesenchymal stem cells to be differentiated into sweat gland-like cells with a sweat gland-like cell phenotype in vitro, and the sweat gland-like cells can form a tubular structure similar to a sweat gland structure in glue, so that the requirement on sweat glands in the process of treating a large-area burn patient can be met.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to an induction culture medium and an induction method for differentiation of stem cells into sweat gland-like cells.
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. The sweat gland is used as an important functional skin accessory, and can regulate body temperature, secrete sweat and discharge partial metabolites of a human body. Through the secretion and evaporation of sweat, sweat glands can remove 25% of the body's heat to keep body temperature relatively constant. The number of sweat glands existing in human body is constant, and the development of the sweat glands is a complex process, and once a large area is damaged, the functional sweat glands cannot be quickly and effectively recovered.
In the case of mild burns, sweat gland-like cells can be completely repaired by differentiation of stem cells by using an undamaged part in the deep part of the sweat gland-like cells as a template, but when the whole skin is deeply burned in a large area, basic conditions for reconstructing a complex structure of the sweat gland by division, proliferation and differentiation of the stem cells are lost, so that even if a wound surface is covered, the function of secreting sweat is lost, so that not only is serious obstacle brought to the physiology and psychology of a patient, but also the life quality of the patient is seriously influenced.
Research on skin tissue repair and functional reconstruction has been a focus of research in the biomedical field. At present, collagenase is utilized to digest skin, sweat gland individuals are dissociated, and sweat gland-like cells can be formed by separation and culture, but the research has some problems, such as difficulty in separation and purification of sweat glands, limited in-vitro culture and passage, limited application and incapability of realizing clinical mass transplantation; meanwhile, a culture medium for in vitro culture of sweat glands is not determined at present, and the in vitro culture effect of the sweat gland-like cells is not ideal.
In addition, it has been found that stem cells co-cultured with sweat gland-like cells can differentiate into sweat gland-like cells. The sweat gland-like cells are subjected to heat shock treatment, the sweat gland-like cells and the mesenchymal stem cells are directly co-cultured after the heat shock, or the sweat gland-like cell culture supernatant and the mesenchymal stem cells are co-cultured after the heat shock, and the mesenchymal stem cells can be differentiated into the cells with the sweat gland phenotype.
Therefore, it is necessary to develop a new induced differentiation medium and establish a novel efficient method for directional differentiation of stem cell sweat gland-like cells.
Disclosure of Invention
The invention aims to provide a technical scheme which can directionally induce and differentiate stem cells, particularly umbilical cord mesenchymal stem cells into sweat gland-like cells. In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides an induction medium for differentiation of stem cells into sweat gland-like cells, wherein the induction medium is a DMEM/F-12 cell culture medium containing the following components:
fetal bovine serum: 3 to 8 percent
Epidermal growth factor: 40-60 ng/mL
Basic fibroblast growth factor: 40-60 ng/mL
Recombinant human ectodermal dysplasia a1 protein: 10-30 ng/mL
Recombinant human Wnt3a protein: 10-30 ng/mL
Bone morphogenetic protein 4: 10-30 ng/mL
Hepatocyte growth factor: 20-30 ng/mL
Triiodothyronine: 1-3 ng/mL
Hemisuccinyl hydrocortisone: 0.1 to 0.2. mu.g/mL
Insulin-transferrin-sodium selenite: 0.5 to 1.5 percent
L-glutamine: 0.5 to 1.5. mu. mol/mL
Penicillin and streptomycin: 80-120U/mL
Among them, DMEM/F-12 cell culture Medium (Gibco Dulbecco's Modified Eagle Medium: F-12) is a widely used basal Medium for promoting the growth of many different mammalian cells, including MDCK, glial cells, fibroblasts, human endothelial cells and rat fibroblasts. The culture medium adopts a 1:1 mixture of a DMEM culture medium and Ham's F-12, combines high-concentration glucose, amino acid and vitamins of the DMEM with more abundant nutrient components in an F-12 culture medium, contains a plurality of trace elements, and has better reliability and consistency when being used for mammalian cell culture.
Further, the induction medium is DMEM/F-12 cell culture medium comprising the following components:
fetal bovine serum: 5 percent of
Epidermal growth factor: 50ng/mL
Basic fibroblast growth factor: 50ng/mL
Recombinant human ectodermal dysplasia a1 protein: 20ng/mL
Recombinant human Wnt3a protein: 20ng/mL
Bone morphogenetic protein 4: 20ng/mL
Hepatocyte growth factor: 25ng/mL
Triiodothyronine: 2ng/mL
Hemisuccinyl hydrocortisone: 0.15. mu.g/mL
Insulin-transferrin-sodium selenite: 1 percent of
L-glutamine: 1. mu. mol/mL
Penicillin and streptomycin: 100U/mL
Further, the ratio of the penicillin to the streptomycin is 1: 2-2: 1, and preferably the ratio of the penicillin to the streptomycin is 1: 1.
Further, the stem cells are mesenchymal stem cells, preferably, the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). Compared with ES cell sources and bone marrow or adipose mesenchymal stem cell sources which are limited or not easy to obtain, the human umbilical cord mesenchymal stem cells (hUC-MSCs) have stronger self-renewal and proliferation capacities, low immunogenicity, convenient material taking, high purity and no virus and tumor cell pollution.
A second aspect of the present invention provides a method of inducing differentiation of stem cells into sweat gland-like cells, comprising the steps of:
s1, obtaining primary cells of the stem cells;
s2, inoculation 5X 104Putting the stem cells in each well in a 6-well cell culture plate, adding 0.5-l mL DMEM/F-12 culture medium containing 10% fetal calf serum in advance, putting the culture plate in a cell culture box, placing the culture plate at 37 ℃, and putting 5% volume CO2Culturing in saturated humidity;
s3, adding the induction medium of claim 1 or 2 after stem cells adhere to the wall, and replacing the induction medium every day; .
S4, after the induced stem cells grow to reach the confluence rate of 80%, absorbing the culture medium, cleaning with a phosphate buffer solution, digesting the cells for 2 minutes by using lmL 0.25.25% pancreatin, stopping the pancreatin by using a serum-containing culture medium, sucking the digested cells into a centrifuge tube, centrifuging at 1200 rpm for 5 minutes, discarding the supernatant, adding the induction culture medium, uniformly mixing cell precipitates, and inoculating the cells into a new culture dish according to the proportion of 1 to 3;
and S5, repeating the step S4, and obtaining the sweat gland-like cells after inducing differentiation for 28 days.
Further, the stem cell is a mesenchymal stem cell, preferably, the mesenchymal stem cell is a human umbilical cord-derived mesenchymal stem cell.
Further, the step of obtaining primary cells of said human umbilical cord-derived mesenchymal stem cells is:
s11, immersing the human umbilical cord in 0.9% sterilized normal saline after obtaining the human umbilical cord;
s12, washing with PBS buffer solution containing 1% penicillin and streptomycin for 3 times, removing bloodstain, fully cleaning umbilical vein lumen, stripping arteriovenous, taking umbilical cord connective tissue, and cutting to 4-5mm3A size tissue mass;
s13, inoculating the tissue block into a 6-hole cell culture plate, adding 0.5-l mL DMEM/F-12 culture medium containing 10% fetal calf serum in advance into the culture plate, placing the culture plate in a cell culture box at 37 ℃ and 5% volume CO2Culturing in saturated humidity;
and S14, changing the liquid for the first time after the cells are cultured for 24 hours, changing the liquid every 2-3 days later, growing the primary cells after the cells are cultured for 12-14 days, and removing tissue blocks after the primary cells grow to be full of 60% to obtain the primary cells.
In some embodiments of the invention, obtaining the primary cell further comprises performing an immunophenotypic adipogenic osteogenic assay on the obtained primary cell to verify that the primary cell has mesenchymal stem cell characteristics.
Further, in step S12, the ratio of penicillin to streptomycin is 1: 2-2: 1.
The invention has the advantages of
Compared with the prior art, the invention has the following beneficial effects:
the sweat gland induction culture medium can effectively induce stem cells, particularly mesenchymal stem cells to be differentiated into sweat gland-like cells with a sweat gland-like cell phenotype in vitro, and the sweat gland-like cells can form a tubular structure similar to a sweat gland structure in glue, so that the requirement on sweat glands in the process of treating a large-area burn patient can be met.
The sweat gland induction culture medium has the advantages of simple culture equipment, simple and quick operation steps, low cost, suitability for induction differentiation of a large number of stem cells and easy popularization when the sweat gland induction culture medium is used for inducing the stem cells, particularly the mesenchymal stem cells to be converted into the sweat gland-like cells.
Drawings
Fig. 1 shows sweat gland-like cells induced by mesenchymal stem cells in example 2 of the present invention.
FIG. 2 shows the result of measuring the sweat gland index of the induced sweat gland-like cells at the mRNA level in example 2 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects solved by the present invention more apparent, the present invention is further described in detail below with reference to the following embodiments.
Examples
The following examples are used herein to demonstrate preferred embodiments of the invention. It will be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function in the invention, and thus can be considered to constitute preferred modes for its practice. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit or scope of the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1 sweat gland-like cell Induction Medium
The embodiment provides a sweat gland-like cell induction culture medium, which is a DMEM/F-12 cell culture medium and further comprises the following components in parts by weight:
fetal Bovine Serum (FBS): 5 percent of
Epidermal growth factor: 50ng/mL
Basic fibroblast growth factor: 50ng/mL
Recombinant human ectodermal dysplasia a1 Protein (echodysplasin a1 Protein): 20ng/mL
Recombinant human Wnt3a protein: 20ng/mL
Bone morphogenetic protein 4: 20ng/mL
Hepatocyte growth factor: 25ng/mL
Triiodothyronine: 2ng/mL
Hemisuccinyl hydrocortisone: 0.15. mu.g/mL
Insulin-transferrin-sodium selenite: 1 percent of
L-glutamine: 1. mu. mol/mL
Penicillin and streptomycin (1: 1): 100U/mL
Example 2 application of sweat gland-like cell induction Medium to induce differentiation of Stem cells into sweat gland-like cells
The stem cells used in this embodiment are human umbilical cord mesenchymal stem cells (hUC-MSCs), and the step of obtaining hUC-MSCs includes:
A. after the human umbilical cord was obtained by the operating table, it was immersed in 0.9% sterile physiological saline and transported aseptically to the laboratory.
B. Taken out of the clean bench, washed 3 times with 1% double antibody (penicillin and streptomycin) in PBS buffer to remove blood ulcer and thoroughly clean umbilical vein lumen, and peeledDropping arteriovenous, collecting umbilical cord connective tissue, and cutting to 4-5mm3Tissue mass of size.
C. Inoculating the tissue block into a 6-well cell culture plate, adding 0.5-l mL DMEM/F-12 medium containing 10% fetal bovine serum in advance, placing the culture plate in a cell culture box at 37 ℃ and 5% volume CO2And culturing in saturated humidity.
D. After the cells are cultured for 24h, the liquid is changed for the first time, then the liquid is changed every 2-3d, and after 12-14d of culture, the primary cells grow out and grow to be full of 60 percent, and then tissue blocks are removed.
E, subculturing hUC-MSCs: placing a culture plate in a cell culture box for culture, wherein the conditions are as follows: temperature 37 ℃ and 5% CO2Saturation humidity, changing the culture medium for the first time after 24h, and then changing the culture medium every 2-3d, observing the cell morphology by a microscope and taking a picture.
The acquired hUC-MSCs are respectively subjected to immunophenotyping, adipogenic osteogenesis and other detections to confirm that the hUC-MSCs have the characteristics of mesenchymal stem cells.
The method for inducing hUC-MSCs with mesenchymal stem cell characteristics to differentiate into sweat gland-like cells by using the sweat gland-like cell induction culture medium in example 1 comprises the following steps:
After B.20h, after the hUC-MSCs are attached to the wall, the sweat gland-like cell induction culture medium prepared in example 1 is added, and then the sweat gland-like cell induction culture medium is replaced every day.
C. After the induced amniotic fluid stem cells grow until the confluence rate is 80%, the culture medium is sucked out, the cells are washed by Phosphate Buffered Saline (PBS), lmL 0.25.25% pancreatin is used for digesting the cells for 2 minutes, the serum-containing culture medium is used for stopping the action of the pancreatin, the digested cells are sucked into a centrifuge tube for centrifugation at 1200 rpm and 5 minutes. Removing supernatant, adding sweat gland-like cell induction culture medium, mixing cell precipitate, and inoculating cells to new culture dish according to the ratio of 1 to 3;
D. repeating the step C, and inducing differentiation for 13 days to obtain sweat gland-like cells as shown in figure 1, wherein the sweat gland-like cells can form a tubular structure similar to a sweat gland structure in the gum.
The method comprises the following steps of (1) detecting sweat gland indexes by using hUC-MSCs as negative controls and human sweat gland-like cells (hSG) as positive controls and using Real time-PCR:
the cells were collected, total RNA was extracted by Trizol extraction, and the reverse transcribed cDNA was diluted 20-fold and used. The PCR system is as follows: 14.95 μ L H2O, 2.5. mu.L of 10 XPCR buffer, 0.75. mu.L of dNTP, 0.75. mu.L of upstream primer, 0.75. mu.L of downstream primer, 0.3. mu.L of Taq enzyme, 5. mu.L of cDNA.
The following primers were used for PCR analysis of sweat gland indicators:
EDA-for：5'TGGAGCCGAGTCCCGCCTTG 3'
EDA-rev：5'CTTCTCCCGGTTCCAATGGCT3'
EDAR-for：5'CCTGTCTCCCTTCCAGCACGC 3'
EDAR-rev：5'GATGTAGAACATGATGATGA 3'
K8-for：5'CAGATGGAGAACGAATTTGTCC 3'
K8-rev：5'TCATATAGCTGCCTGAGGAAGTTG 3'
CEA-for：5'TGGGTAAACAATCAGAGCCTCC 3'
CEA-rev：5'TGGGTTCTGGGTTTCACATTTG 3'
GAPDH-for：5’CTGGGCTACACTGAGCACC3’
GAPDH-rev：5’GTGGTCGTTGAGGGCAATG3’
the PCR reaction conditions were as follows:
pre-denaturation at 95 deg.C for 10 min; denaturation at 95 ℃ for 35 s; annealing, the Tm value (EDA: 58 ℃, EDAR: 61 ℃, K8: 59 ℃, CEA: 58 ℃ and GAPDH: 58 ℃) corresponding to each primer; extension at 72 ℃ for 35 s; reacting for 35 cycles, extending for 10min at 72 ℃, and storing for later use at 4 ℃.
The Real time-PCR results are shown in FIG. 2, and it can be seen from FIG. 2 that hUC-MSCs gradually express sweat gland-like cell indexes EDA, EDAR, K8 and CEA in the process of induction, and gradually approach sweat gland-like cells in the process of differentiation.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Claims (10)
1. An induction medium for differentiation of stem cells into sweat gland-like cells, wherein the induction medium is a DMEM/F-12 cell culture medium comprising the following components:
fetal bovine serum: 3 to 8 percent
Epidermal growth factor: 40-60 ng/mL
Basic fibroblast growth factor: 40-60 ng/mL
Recombinant human ectodermal dysplasia a1 protein: 10-30 ng/mL
Recombinant human Wnt3a protein: 10-30 ng/mL
Bone morphogenetic protein 4: 10-30 ng/mL
Hepatocyte growth factor: 20-30 ng/mL
Triiodothyronine: 1-3 ng/mL
Hemisuccinyl hydrocortisone: 0.1 to 0.2. mu.g/mL
Insulin-transferrin-sodium selenite: 0.5 to 1.5 percent
L-glutamine: 0.5 to 1.5. mu. mol/mL
Penicillin and streptomycin: 80-120U/mL.
2. The induction medium according to claim 1, wherein the induction medium is DMEM/F-12 cell culture medium comprising the following components:
fetal bovine serum: 5 percent of
Epidermal growth factor: 50ng/mL
Basic fibroblast growth factor: 50ng/mL
Recombinant human ectodermal dysplasia a1 protein: 20ng/mL
Recombinant human Wnt3a protein: 20ng/mL
Bone morphogenetic protein 4: 20ng/mL
Hepatocyte growth factor: 25ng/mL
Triiodothyronine: 2ng/mL
Hemisuccinyl hydrocortisone: 0.15. mu.g/mL
Insulin-transferrin-sodium selenite: 1 percent of
L-glutamine: 1. mu. mol/mL
Penicillin and streptomycin: 100U/mL.
3. The induction medium according to claim 1 or 2, wherein the ratio of penicillin to streptomycin is 1:2 to 2: 1.
4. The induction medium according to claim 1 or 2, wherein the stem cells are mesenchymal stem cells.
5. A method of inducing differentiation of stem cells into sweat gland-like cells, comprising the steps of:
s1, obtaining primary cells of the stem cells;
s2, inoculation 5X 104Putting the stem cells in each well in a 6-well cell culture plate, adding 0.5-lmL DMEM/F-12 culture medium containing 10% fetal calf serum in advance, placing the culture plate in a cell culture box at 37 ℃ and 5% volume CO2Culturing in saturated humidity;
s3, adding the induction medium of claim 1 or 2 after stem cells adhere to the wall, and replacing the induction medium every day; .
S4, after the induced stem cells grow to reach the confluence rate of 80%, absorbing the culture medium, cleaning with a phosphate buffer solution, digesting the cells for 2 minutes by using lmL 0.25.25% pancreatin, stopping the pancreatin by using a serum-containing culture medium, sucking the digested cells into a centrifuge tube, centrifuging at 1200 rpm for 5 minutes, discarding the supernatant, adding the induction culture medium, uniformly mixing cell precipitates, and inoculating the cells into a new culture dish according to the proportion of 1 to 3;
and S5, repeating the step S4, and obtaining the sweat gland-like cells after inducing differentiation for 28 days.
6. The method of claim 4, wherein the stem cell is a mesenchymal stem cell.
7. The method of claim 5, wherein the mesenchymal stem cells are human umbilical cord-derived mesenchymal stem cells.
8. The method of claim 6, wherein the step of obtaining the primary cells of human umbilical cord-derived mesenchymal stem cells is:
s11, immersing the human umbilical cord in 0.9% sterilized normal saline after obtaining the human umbilical cord;
s12, washing with PBS buffer solution containing 1% penicillin and streptomycin for 3 times, removing bloodstain, fully cleaning umbilical vein lumen, stripping arteriovenous, taking umbilical cord connective tissue, and cutting to 4-5mm3A size tissue mass;
s13, inoculating the tissue block into 6-hole cell culture plate, adding 0.5-lmL DMEM/F-12 culture medium containing 10% fetal calf serum in advance, placing the culture plate in a cell culture box at 37 ℃ and 5% volume CO2Culturing in saturated humidity;
and S14, changing the liquid for the first time after the cells are cultured for 24 hours, changing the liquid every 2-3 days later, growing the primary cells after the cells are cultured for 12-14 days, and removing tissue blocks after the primary cells grow to be full of 60% to obtain the primary cells.
9. The method of claim 8, wherein obtaining the primary cells further comprises performing an immunophenotypic adipogenic osteogenic assay on the obtained primary cells to verify that the primary cells have mesenchymal stem cell characteristics.
10. The method of claim 8, wherein the ratio of penicillin and streptomycin in step S12 is 1: 2-2: 1.
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