The Mechanism Of DNA Methylation During The Induction Of Sweat Gland Like Cells From Human Bone Marrow-mesenchymal Stem Cells

Objective:(1) To optimize the method of isolating human eccrine sweat gland cells and to get primary human sweat glands efficiently.(2) To establish the induction system that promoted human bone marrow mesenchymal stem cells(BM-MSCs) to differentiate into sweat glands like cells(SGLCs), and investigate the change on morphology、cytological markers、related gene expression.(3) To explore the effect and mechanism of DNA methylation during this induction process and to found the expression and regulation of target genes. Methods:(1) The fresh and normal skin tissue was cut into pieces of microskin. The three groups(mixture of Trypsin-EDTA and Collagenase 、 Trypsin-EDTA buffer and Collagenase buffer) were experimented for disgeting microskin to release sweat glands. The morphology and growth of three groups was observed after transfering by micropipitter. The proliferation index was detected by flow cytometry after 9 days. The identification of cultured cells was performed by immunocytochemical staining, and the cell surface markers of sweat glands used were cytokeratin 7 and CEA.(2) Cell lines of human BM-MSCs were brought from company. BM-MSCs were cultured and expended in vitro, and exposed its potential differentiation ability by Adipogenic and Osteogenic differentiation. The induced system had been established. We picked up P3 BM-MSCs and heat-shocked sweat gland cells into the transwell chamber to co-culture, while adding cell factors. The change of morphology was observed at the 3th、6th、9th days, respectively, and positive control group was sweat gland cells, respectively. The induced cells were examined by immunecytochemistry, RT-PCR and Western Blotting. The cell surface markers used were cytokeratin 7 and CEA.(3) Illumina Human methylation 450 genome-wide Beadchip was applied for the differential methylation change during the induced-differentiation process. Bioinformatic analysis for SGLCs was done at several aspects, such as, Diff-site Screening, Hierarchical Cluster Analysis, Gene Ontology, KEGG pathway, Biology Funcation Annotation, Protein-Protein interaction networks. Bioinformatic analysis of the Beadchip combined with revelant reseaches and pre-experiment in our team, several candidate genes(HDAC4、PAX6、MMP1、EDA et.al) were aimed.The experimental results were verified preliminary by Methylation Specific PCR. The specific methylation profile of HDAC4 were validated through MALDI-TOF Mass Array further. Finally, recombinant plasmid was transfected into BM-MSCs. The expression of related gene and the change of phenotype in transfected cells were detected. Results:(1) After digesting 2 h, there were lots of dissociated sweat glands emerging in A group. The emergence of dissociated sweat glands in collagenase-Ⅱ group required at least 6 h. The sweat glands cells in group A and B adhered tightly and grew very well. The proliferation index detected by flow cytometry were 18±4% and 17±6% in group A and B, respectively. The results of immune-cytochemical staining showed that there were no significant difference in CEA and CK7 expresion between two groups.(2) It also showed that BM-MSCs can differentiate into adipocytes and osteocytes. The BM-MSCs which had morphology change were scarce at the 3th day. The phenotype of SGLCs was partly obtained after 9 days of co-culture. The cells became irregular in shape and focused in the center. The surface antigens of sweat glands, such as cytokeratin 7, CEA were detected in SLGCs by immunecytochemistry, RTPCR, and Western Blotting.(3) Illumina 450 K genome-wide Beadchip showed that the significantly different sites were 693. It was showed that a total of 437 genes were differences in methylation level between before and after induction(97 up-methylation genes and 350 downmethylation genes).The coverage throughout gene regions, was mainly on CpG islands, N_Shore,Gene body and TSS 200. The Gene Ontology and the KEGG pathway showed that predicted genes were mainly involved in a variety of biological pathyways, such as cell fate determination, signal transduction involved in regulation of gene expression, transcription corepressor activity, ectodermal development et.al. The results of MSP and Mass Array displayed methylation level of HDAC4 were decreased obviously.Meanwhile, the detection of Beadchip demonstrated the similar tendency. The result of immunocytochemistry and RT-PCR showed that the transfected BM-MSCs by recombinant plasmid could improve the transcription of CEA. Conclusion:(1) Trypsin-EDTA combined with collagenase-Ⅱcan shorten the time of isolating sweat gland cells and there was no significant difference in cell proliferation and surface marks.(2) The co-culture system which consisted of BM-MSCs and heated-shock SGCs provided an advantageous micro-environment, and obtained SGLCs successfully.(3) As the important modification in epigenetic, DNA methylation might participate in and regulate the induced process.(4) The results pointed to the suspect that HDAC4 might regulate the transcription and endocytosis of CEA, and conduct the induced-differentation process.