摘要
Objective To express mouse peroxisome proliferator activated receptor γ2 (mPPARγ2) in NIH3T3 fibroblasts mediated by the recombinant retrovirus and study its function.Methods The mPPARγ2 gene was subcloned into retrovirus vector pGCEN to generate the recombinant pGCEN/mPPARγ2. Then it was packaged into PA317 cells and selected with G418. Viral supernatants were harvested and then used to infect NIH3T3 fibroblasts. PPARγ activator 5,8,11,14-eicosatetraynoic acid (ETYA) was used to induce the mPPARγ2-expressing NIH3T3 cells into adipocyte differentiation.Results The recombinant retrovirus pGCEN/mPPARγ2 was constructed, and the higher titers of the viral supernatants were obtained. mPPARγ2 was expressed in NIH3T3 cells mediated by the recombinant retrovirus. Lipid accumulation obviously existed in these induced adipocytes which morphologically resembled mature adipocytes in vivo and expressed tissue specific adipocyte P2 (AP2) and Leptin genes.Conclusions An adipocyte differentiation model in vitro was successfully established. The work is the basis for further research on the molecular mechanism of adipocyte differentiation induced by PPARγ2.
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 。
基金
ThisstudywassupportedbyagrantfromtheShanghai UnileverResearch&DevelopmentFund (No 980 7)
