| Object:(1) To observe the morphological changes and to explore the cytokeratin expression of murine sweat gland cells during the developmental process.(2) To retrieval the biological difference of murine epidermal stem cells at E12.5 and mature sweat gland cells at P5 in the same position, as well as, to validate the coincidence between epidermal stem cells in foot and in back at the same stage(E12.5).(3) To screen and identify the key determinant of the differentiation switch of epidermal progenitors to sweat gland cells.(4) To explore effects of the key determinant during the process of the differentiation switch of epidermal progenitors to sweat gland cells, and to lay the foundation for the regeneration of sweat glands.Materials and methods:(1) We isolated and cultured primary sweat gland cells from murine foot pads at different stages(E12.5, E17.5, P0.5, P5 and P28), as well as observed morphological changes of the eccrine sweat gland cell during the developmental process. Then we detected the cytoketain expression of murine eccrine sweat gland cells by immunofluorescence and RT-PCR at different stages.(2) We isolated and cultured primary epidermal stem cells and sweat gland cells from E12.5 and P5 murine foot pads, as well as observed morphological distinction. Then we detected the structure of skin in different time points and locations by hematoxylin and eosin stain. Meanwhile, we observed the contrast of cytoketain expression of murine epidermal stem cells and eccrine sweat gland cells by immunofluorescence. At equal pace, we detected the coincidence between epidermal stem cells in foot and in back at the same stage(E12.5) by hematoxylin and eosin stain and immunofluorescence.(3) We isolated and cultured primary epidermal stem cells and sweat gland cells from E12.5 murine back epidermis and P5 murine foot pads, extracted total RNA by Trizol, then identified the key determinant of the differentiation switch of epidermal progenitors to sweat gland cells by Hi Seq2500 and bioinformatics analysis. As well, we validated molecular biology by Western-blot and RT-PCR.(4) We conducted Fox-c1 and Irf-6 related chronic virus to infect the murine epidermal stem cells, and divided into four groups, Control group, Fox-c1 group, Irf-6 group and Fox-c1+Irf-6(F+I) group. CCK-8 kit was used to detect the cell proliferation activity. Meanwhile, we observed changes of morphology and keratin expression of the transfected cells after 14 days by immunfluorescence and Q-PCR. Construction of the deep two degree burn toe mouse model, transfected cells were injected into the burn, after 11 days of testing. The morphology of sweat gland cells in vivo was observed by hematoxylin and eosin stain. The expression of keratin in sweat gland cells was detected by immunfluorescence after different transfection group were injected into the mice. At last, we observed the function of sweat gland cells by iodine starch test.Results:(1) Cellular results showed that the morphology of sweat gland progenitors was similar to epidermal stem cells at E12.5 and E17.5, the duct part and secretary part of sweat gland were formed gradually from P0.5 to P28. Cellular immunofluorescense results indicated that K8 and K19 expression were progressively increased during developmental process of mice eccrine sweat gland cells, K14 was positive expressed only at E12.5 and E17.5, K10 expression was positive after birth. Histological results revealed that K8 and K19 positive expressed cells localized in secretary portion of glands, K10 positive expressed cells localized in the duct portion of glands, K14 positive expressed cells localized in myoepithelium portion of glands.(2) Cellular results showed the morphology of mouse epidermal stem cells at E12.5 present long spindle shape, and the morphology of sweat gland cells at P5 appeared duct department. HE staining results showed that the mice foot pads at E12.5 without sweat gland structure, and the foot pads of P5 present sweat gland structure. Immunofluoresence results show that K5 and K14 were expressed strongly in epidermal stem cells, and K8?K18 were expressed highly in sweat gland cells. No matter HE staining results and Immunofluoresence results showed that the mice back skin tissue and foot pads at E12.5 are concord.(3) Bioinformatics analysis screened Fox-c1 and Irf-6 are the key determinant of the differentiation switch of epidermal progenitors to sweat gland cells. RT-PCR and Western-blot results showed Fox-c1 and Irf-6 were expressed highly in sweat gland cells than in epidermal stem cells.(4) CCK-8 kit detecting result showed that the change of cellular activity. Cellular results showed the morphology of sweat gland cell-like after epidermal stem cells were transfected 14 days. Immunofluoresence and Q-PCR results showed that whether K5/K14 or K8/K18 was expressed strongly in tranfected cells. HE staining of murine foot pads were infected with transfected cells after 14 days showed that there was no sweat gland-like structure in control group and Irf-6 group, and there were some sweat gland-like structures in Fox-c1 group and Fox-c1+Irf-6(F+I) group. Tissue immunofluoresence results in vivo were in common with cellular immunofluoresence results in vitro. Iodine starch test showed that results of control group and Irf-6 group were negative, as well as results of Fox-c1 group and Fox-c1+Irf-6(F+I) group were positive.Conclusion:(1) The result of this study has demonstrated the biological markers of mouse sweat gland cells, and provides a useful tool for the study of sweat gland regeneration.(2) The result of this study has discovered the distinct of morphology and keratin expression between murine epidermal stem cells and sweat gland cells in foot pads at different stages(E12.5 and P5), and the coincidence between epidermal stem cells in foot and in back at the same stage(E12.5).(3) The result of this study has screened Fox-c1 and Irf-6 are the key determinant of the differentiation switch of epidermal progenitors to sweat gland cells.(4) The result of this study has explored Fox-c1 and Irf-6 can promote the differentiation switch of epidermal progenitors to sweat gland cells, and provided the foundation of sweat glands regeneration. |
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