PPARγ基因RNA干扰慢病毒载体构建及干扰Leydig细胞株建立

Construction of a Lentiviral Vector of RNA Interference against PPARγ Gene and the Establishment of Leydig Cell Line

目的:通过靶向过氧化物酶体增殖因子活化受体PPARγ基因阻断,建立稳定干扰Leydig细胞株TM3。方法:采用慢病毒四质粒包装系统构建特异靶向小鼠PPARγ基因的RNA干扰(RNA interference,RNAi)慢病毒载体,用以建立PPARγ基因稳定沉默的Leydig细胞株TM3。结果:经测序证实,成功构建编码表达PPARγ短发夹结构RNA(sh-PPARγ)的慢病毒载体LV3-sh-PPARγ及对照载体LV3-Control。病毒载体转导Leydig细胞株TM3后,荧光显微镜证实细胞转染效率>90%,Real-time PCR测定实验组TM3细胞PPARγ的mRNA表达比未处理细胞下降70%(P<0.05),对照组与未处理细胞差异无显著性(P>0.05)。Western blot显示实验组PPARγ蛋白表达量比未处理细胞明显下降(P<0.05),对照组与未处理细胞差异无显著性(P>0.05)。结论:成功构建特异靶向小鼠PPARγ基因的RNAi慢病毒载体LV3-sh-PPARγ及对照载体LV3-Control,并成功建立PPARγ表达稳定干扰的小鼠Leydig细胞株TM3,为PPARγ生殖毒性研究提供了新的细胞模型。

Ojective： To investigate the targeting peroxisome proliferator-activated receptor PPARγ gene blocked the establishment of a stable interference Leydig cell lines of TM3. Methods： Four plasmid lentiviral packaging systems were used to build the specific targeting of PPARγ gene in mice by RNA interference （RNAi） lentiviral vector, and Leydig cell lines （ TM3 ） was used to establish stable silencing of the PPARγ gene. Results： DNA sequencing confirmed that encoding expression of the lentiviral vector LV3-sh-PPAR3, PPARγ short hairpin RNA （sh-PPARγ） and control vector LV3-Control were successfully constructed. After the transduction, highly efficient transfection （ 〉 90% ） of lentivirus in TM3 cells was observed by fluorescence microscopy. Quantitative Real-time PCR showed that LV3-sh-PPARγ lentivirus reduced the PPARγ mRNA expression by about 70% in the experimental TM3 cells as compared with that in untreated cells （ P 〈 0. 05 ）, while no significant differences was found between control group and untreated cells group （ P 〉 0. 05 ）. Western blot revealed that the PPARγ protein expression decreased significantly in LV3-sh-PPARγ treated cells group （ P 〈 0. 05 ） , compared with that in untreated cells, and no significant differences were found between control group and untreated cells group （ P 〉 0. 05 ）. Conclusion： The specific targeting of PPARγ gene in mice RNAi lentiviral vector LV3-sh-PPARγ and vector LV3-Control are successfully constructed, and the expression of PPARγ steady interference in mice Leydig cell line of TMa is successfully established, and is provided a new cell model for PPARγ/in reproductive toxicity study.