A microRNA induction method for inducing the transdifferentiation of BM-MSCs into sweat gland cells in vitro
A sweat gland cell and transdifferentiation technology, applied in the field of human sweat gland regeneration research, can solve the problems of induced microenvironment system instability, cell fusion, easy mixing of cells, and inability to pass passage
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Embodiment 1B
[0016] Isolation and identification of embodiment 1BM-MSCs
[0017] 1. Isolation and culture of BM-MSCs: take about 2ml of the ilium bone marrow of normal volunteers under sterile conditions, adopt the direct adherence method, and contain 10% fetal bovine serum, penicillin (100U / ml) and streptomycin (100μg / ml) of DMEM as the culture medium, cultivated in an incubator at 37°C and 5% CO2, replaced the medium after 24 hours, discarded the non-adherent cells, and changed the medium once every 2-3 days thereafter. When the cells reached 80% confluence, they were digested with 0.125% trypsin and subcultured ( figure 1 A-C).
[0018] 2. Immunocytochemical test results: take cell slides, fix with pre-cooled formaldehyde acetone mixture (volume ratio 1:1)) for 30 min, and perform CD44 detection according to the instructions of the two-step immunohistochemical detection kit. The detection results showed that CD44 was strongly positively expressed in BM-MSCs ( figure 1 D).
Embodiment 2
[0019] Example 2 Has-miR-138-5p inhibitor transfection of BM-MSCs and flow sorting
[0020] microRNA transfection into BM-MSCs: add the chemically synthesized Has-miR-138-5p inhibitor and negative control of microRNA inhibitors to DEPC water and dilute to a concentration of 20uM. The third generation MSCs with 3×10 5 / ml density inoculation in T25cm 2 cultured in a sterile plastic culture flask until the cells reached 60% confluency. Take a disposable sterile enzyme-free 1.5ml EP tube, first add 500ul Opti-MEM I, then add 30ul (600pmol) of the chemically synthesized microRNA mimics (inhibitor) diluted above, and mix gently; Enzyme 1.5ml EP tube, first add 500ul Opti-MEM I, then add 15ul Lipo2000, mix gently, let stand at room temperature for 5min; use sterile enzyme-free tips to dilute chemically synthesized microRNAs and Lipo 2000 in the above 1.5ml EP tube Mix slowly and gently, let stand at room temperature, and incubate for 20 minutes (to promote the formation of microR...
Embodiment 3
[0021] Example 3 microRNAs transfection BM-MSCs 7d detection of sweat gland surface marker protein (cell immunochemical staining)
[0022] After microRNAs induction for 7 days, fix with 4% paraformaldehyde for 15-20 min, permeabilize with 0.2% Triton X-100 for 10 min, add 3% H 2 o 2 Incubate at room temperature for about 10 minutes to remove endogenous peroxidase, add CEA, CK19, CK7, CK8 and CK14 primary antibodies diluted in a certain proportion, overnight at 4°C, rinse with PBS for 3 times, add HRP-labeled anti-mouse and anti-rabbit Secondary antibody, covered at room temperature for 30min, rinsed with PBS 3 times, developed with DAB, counterstained with hematoxylin for nuclei. The results showed that some cells in the has-miR-138-5p inhibitor transfection group had positive expression of sweat gland marker proteins CEA, CK19, CK7, CK8 and CK14 (brown yellow) ( image 3 A, C, E, G and I), but no positive brown staining cells were found in the negative control group ( ima...
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