| Objectives: Being able to simultaneously express all genes of germline, endoderm, mesoderm and ectoderm, bone marrow mesenchymal stem Cells ( MSCs ) are considered to have the potential to differentiate to other tissues and cell lines. Except for the differentiation to bone tissue, cartilage tissue, nerve tissue, mussle tissue and adipose tissue, recently MSCs are reported to also have the potential of differentiation to the epidermic cells and vascular endothelial cells of granulation tissue under the circumstance of wound. But the mechanism by which the MSCs differentiate remains unclear. Simultaneously, as stem cells derived from adult mammal, MSCs are easy to be isolated and cultured, they play more and more important roles in the field of tissue engineering and wound repair. Since MSCs have the characteristic of multiple differentiation potential and are able to locate into bone marrow and survive for a long time by self renewing after being injected, many target genes mediated by retrovirus can be integrated into the genome DNA of MSCs and be expressed chronically, MSCs can be used in gene therapy as a type of ideal target cell.Serum derived from burned mammal, which is a compound that is composed of many active components, changes its elements with the time after burn injury. These serum components involves complement, intercellular adhesion molecule, interleukin, colony stimulating factor, tumor necrosis factor, interferon. It is possible that many other unknown factors that can affect the gene expression ofMSCs have been involved. When added to the MSCs culture system, serum derived from burned rats can partially affect the status of MSCs. Components contained in the serum may be the major factors that induced the change of gene expression of MSCs.Based on the fact that MSCs are easy to be isolated and cultured in vitro, with the methods of cell culture, immunocytochemistry, flow cytometer and gene chip, the object of this study is to observe the effects of serum derived from burned rats on the changes of biological behavior and gene expression of MSCs and to dicuss the relationship of the effects with burn injury insult underlying the mechanisms of MSCs' particapting the process of wound repair, repairing the wound of burn injury with a small volumn of quickly proliferative MSCs .Methods: Bone marrow was extracted from sacrificed Wistar rats and MSCs were then incubated in F-12 medium in the presence of fetal calf serum, normal rat serum, serum derived from burned rats for 3 days and 14 days after burn injury. The growth curves of MSCs were investigated with methods of PI staining and MTT, cell cycle, apoptosis rate and contents of DNA of MSCs were investigated with flow cytometer.Expression of Pan Cytokeratin and Factor VIII related antigen of MSCs were detected with immunocytochemical method and flow cytometry. The procedure of immunocytochemical method was strictly followed the manufacturer's instruction. Negative controls without primary antibodies were simultaneously detected. Stained sections were observed with light microscope. The cellular plasm of positive cells had been stained into buffy.Total RNA was isolated with TRIzol methods from MSCs incubated with F-12 medium in the presence of serum derived from normal rats and burned rats for 24 hours and 72 hours, respectively. The mRNA from normal rats and burned rats were reversely transcripted into cDNA, and labelled with cy3-dUTP and cy5-dUTP respectively. The difference of gene expression between two groups of MSCs were then detected with oligonucleotide micorarrays each of which contains 5705 genes .The results were statistically analysed with STATA software software.Results: The growth curves of cells incubated with serum from burned rats were significantly different from that of cells incubated with serum from normal rats. The doubling time of cells incubated with serum from burned rats were significantly shortened compared with that of cells incubated with... |
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