| Summary: | 碩士 === 慈濟大學 === 人類遺傳研究所 === 93 === The causes for obesity were complicated. There are many factors contribute to the development of obesity: genetics, environment, and their interactions. Obesity usually goes along with the increases in adipose tissue. In this study, we focused on the analysis of adipocyte-specific genes expression during preadipocyte differentiation into adipocyte.
We used two cell line models, human marrow-derived multipotent mesenchymal stem cells and mouse 3T3-L1 fibroblast cells. After treating with differentiation promoted drugs, we collected the cells at different time points from day 0 to day 12. The total RNA was purified and transcripted to cDNA. The expression of PPAR-g, Adiponectin, Leptin, and Resistin genes were analyzed by qPCR analyses. All of these four proteins were adipocyte specifically secreted and deeply involved in fat and glucose metabolism of body. We also constructed and assembled these four genes and some control genes primer sequences into pyT&A vectors which called pIChuman and pICmouse as the standard for gene expression experiments.
During the differentiation into the adipocyte from the human marrow-derived multipotent mesenchymal stem cells. Adiponectin expression started to appear at day 4（9 copies per 100 cells）and the gradually increased to day 13（290 copies per 100 cells）. Leptin was detected until day 13（10 copies per 100 cells）, and Resistin was not detected in any time shifts. By the real-time PCR analysis, we were able to detect PPAR-g from day 0.5（200 copies per 100 cells）to day 13 and the day 13 had the highest expression（8,600 copies per 100 cells）. We also analyzed various human marker gene expression by real-time (Taqman probe) PCR and find the expression of Nestin, Extrogen receptor, HNF4a, b-globin and Flk 1 had different expression in early differentiation stage （day 0.5）and not or weakly expression in late differentiation stage. It showed that the human marrow-derived multipotent mesenchymal stem cells followed differentiation pathway.
During the differentiation into adipocyte from the mouse 3T3-L1 fibroblast cell, PPAR-g was expressed first, although weakly, at the day 1（2 copies per 100 cells）and was then gradually increased to 500 copies per 100 cells at day 12 . Adiponectin and Resistin were expressed at day 4（50 and 2 copies per 100 cells）,and continues increase follow the differentiation time （2100 and 100 copies per 100 cells）whereas Leptin was expressed very weakly and was detected only at day 4（2 copies per 100 cells）.
For study the gene product structure, function, and generation antibody from Adiponectin, Leptin and Resistin. We isolated the full-length cDNA from human these three genes. Cloned them into expression vectors（pQE-Tri）.These three proteins had 8 His in C-terminal. We successful purified these three proteins by Ni-affinity column.
During the expression of adipocyte-specific genes in differentiation, we found that genes expression pattern were consistent with the cell morphology change. Furthermore, we found that the rosiglitazone can significant improve the efficiency of adipocyte differentiation（from 10% to 60-70%）. We suggest add the rosiglitazone as the routines differentiation drug.
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