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目的 通过应用重组逆转录病毒介导小鼠过氧化物酶体增殖体激活受体γ2(mPPARγ2 )基因整合入NIH3T3细胞基因组中并进行表达。方法 从经测序证实含正确序列mPPARγ2的重组质粒pcDNA3/mPPARγ2 中 ,双酶切下约 1.5Kb的mPPARγ2 全长cDNA编码序列 ,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 。pGCEN/mPPARγ2 及pGCEN经LipofectAMINE感染病毒包装细胞系PA317细胞 ,通过筛选PA317细胞G418抗性克隆 ,收集病毒上清 ,然后用其感染靶细胞NIH3T3细胞 ,用免疫荧光染色及Western印迹方法鉴定mPPARγ2 在NIH3T3细胞中的表达情况。结果 构建了含mPPARγ2 全长cDNA重组逆转录病毒载体 ,获得了滴度分别为 5×10 4 CFU/ml和 6× 10 5CFU/ml的pGCEN/mPPARγ2 及pGCEN的病毒上清。经鉴定pGCEN/mPPARγ2 能有效地感染靶细胞NIH3T3细胞并表达mPPARγ2 。结论 本研究结果为在体外建立脂肪细胞分化模型及为进一步研究PPARγ2 在诱导脂肪细胞分化中的分子机制奠定了基础
OBJECTIVE: To synthesize and express the peroxisome proliferator activated receptor γ2 (mPPARγ2) gene in NIH3T3 cells by using recombinant retrovirus. Methods The mPPARγ2 full-length cDNA encoding about 1.5 kb was digested by restriction endonucleases (pcDNA3 / mPPARγ2) containing the correct sequence of mPPARγ2 and subcloned into retroviral vector pGCEN to construct recombinant retroviral vector pGCEN / mPPARγ2 . pGCEN / mPPARγ2 and pGCEN were transfected into PA317 cells by LipofectAMINE, and the supernatant of G418 was collected by screening PA317 cells. The infected cells were infected with NIH3T3 cells. Immunofluorescence staining and Western blotting were used to identify the expression of mPPARγ2 in NIH3T3 Cell expression. Results The mPPARγ2 full-length cDNA recombinant retroviral vector was constructed and the virus supernatants of pGCEN / mPPARγ2 and pGCEN with titer of 5 × 10 4 CFU / ml and 6 × 10 5 CFU / ml were obtained. It has been identified that pGCEN / mPPARγ2 effectively infects target cell NIH3T3 cells and expresses mPPARγ2. Conclusion The results of this study lay the foundation for establishing adipocyte differentiation model in vitro and further studying the molecular mechanism of PPARγ2 in inducing adipocyte differentiation