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目的 应用重组逆转录病毒载体介导小鼠过氧化物酶体增殖体激活的受体γ2 (mPPARγ2 )基因在NIH3T3成纤维细胞中表达 ,并进一步研究其功能。方法 从经荧光测序证实含正确mPPARγ2cDNA序列的重组质粒pcDNA3/mPPARγ2中 ,双酶切下mPPARγ2全长cDNA序列 ,亚克隆入逆转录病毒载体pGCEN中 ,构建重组逆转录病毒载体pGCEN/mPPARγ2。用PA317细胞对pGCEN/mPPARγ2及pGCEN进行包装 ,并用G4 18进行PA317细胞抗性克隆筛选 ,收集病毒上清 ,感染靶细胞NIH3T3成纤维细胞。表达mPPARγ2的NIH3T3成纤维细胞在含PPARγ激活物 5 ,8,11,14-二十碳四烯酸(ETYA)等分化介质中培养 ,可被诱导向脂肪细胞分化。结果 构建了含mPPARγ2全长cDNA重组逆转录病毒载体pGCEN/mPPARγ2 ,获得了较高pGCEN/mPPARγ2及pGCEN的逆转录病毒上清。重组逆转录病毒载体可介导mPPARγ2在NIHT3T3成纤维细胞胞核中表达。油红O染色证实 ,表达mPPARγ2的NIHT3T3成纤维细胞在分化介质中培养分化 10天后 ,胞浆中明显积聚了较多的中性脂肪 ,其细胞形态也与体内成熟脂肪细胞相似 ,而且这些细胞表达脂肪细胞特异性标志基因如AP2和leptin。结论 本研究在体外成功地建立了由PPARγ2诱导NIH3T3成纤维细胞向脂肪细胞分化的模型 ,为进一步研究PPARγ2诱导脂肪细
Objective To investigate the expression of mPPARγ2 gene in mouse NIH3T3 fibroblasts induced by peroxisome proliferator-activated protein (rPVARγ2) using recombinant retroviral vector and further investigate its function. Methods The full length cDNA sequence of mPPARγ2 was cloned from the recombinant plasmid pcDNA3 / mPPARγ2 with the correct mPPARγ2 cDNA sequence by fluorescence and subcloned into the retroviral vector pGCEN to construct recombinant retroviral vector pGCEN / mPPARγ2. PGCEN / mPPARγ2 and pGCEN were packaged with PA317 cells and cloned by G418 for PA317 cell resistance. The virus supernatants were collected and infected with NIH3T3 fibroblasts. NIH3T3 fibroblasts expressing mPPARγ2 are cultured in differentiation media containing PPARγ activators 5, 8, 11, 14-eicosatetraenoic acid (ETYA) and can be induced to differentiate into adipocytes. Results The recombinant retroviral vector pGCEN / mPPARγ2 containing mPPARγ2 was constructed and the retroviral supernatants with higher pGCEN / mPPARγ2 and pGCEN were obtained. Recombinant retroviral vector can mediate the expression of mPPARγ2 in the nucleus of NIHT3T3 fibroblasts. Oil red O staining confirmed that NIHT3T3 fibroblasts expressing mPPARγ2 accumulated significantly more neutral fat in the cytoplasm 10 days after they were differentiated in differentiation medium, and their cell morphology was also similar to that of mature adipocytes in vivo, and these cells expressed Adipocyte-specific marker genes such as AP2 and leptin. Conclusion This study successfully established in vitro NIH3T3 induced by PPARγ2 fibroblasts differentiate into adipocytes, in order to further study the PPARγ2 induced fat thin