3种介导猪NR2F2基因转染PK15细胞方法的比较研究

试验旨在研究3种转染方法对猪肾上皮细胞（PK15）的转染效率,为以PK15细胞为模型研究外源基因的功能提供参考。本研究以PK15细胞为研究对象,用脂质体、电穿孔、慢病毒3种转染方法转染后,在荧光显微镜下观察绿色荧光蛋白的表达,采用实时荧光定量PCR检测EGFP和NR2F2基因的表达情况,采用CCK-8检测细胞存活率,进而比较3种方法的转染效果。结果显示,转染PK15细胞后,电穿孔和慢病毒方法转染效率极显著高于脂质体（P<0.01）,但电穿孔方法和慢病毒方法之间差异不显著（P>0.05）;EGFP和NR2F2基因的实时荧光定量PCR结果显示慢病毒方法效果最好,脂质体方法较差,与细胞转染效率基本一致;CCK-8结果显示,电穿孔转染后细胞存活率最低,极显著低于对照组（P<0.01）,脂质体方法显著低于对照组（P<0.05）,慢病毒方法与对照组间差异不显著（P>0.05）。综合考虑转染效率及细胞活性,本研究认为慢病毒转染方法最适合转染PK15细胞,为今后高效转染PK15细胞提供了理想的方法。

Comparative Study on Three Methods of Mediated Porcine NR2F2 Gene Transfection into PK15 Cells

The aim of this study was to compare the efficiency of transfected pig kidney epithelial cells (PK15) by three transfected methods,so as to lay the foundation for studying the function of foreign genes with PK15 cells as a model.In this study,PK15 cells as research subject were transfected with liposome,electroporation and lentivirus methods.The transfected efficiency was evaluated based on the expression of green fluorescent protein observed by fluorescence microscope,the expressions of EGFP and NR2F2 genes were detected by Real-time quantitative PCR and the cell survival rate was detected by CCK-8 reagent.The results showed that the transfected efficiency of PK15 cells transfected with electroporation and lentivirus methods was extremely significantly higher than that of liposomes (P<0.01),whereas the difference between electroporation and lentivirus methods was not significant (P>0.05).The Real-time quantitative PCR results showed that the expressions of EGFP and NR2F2 genes in PK15 cell transfected with lentivirus method were the highest and the lowest with liposome method,which was in accordance with the transfected efficiency.CCK-8 results showed that the cell survival rate transfected by electransfection was the lowest,which was extremely significantly lower than that of control group (P<0.01).The cell survival rate transfected with the liposome was significantly lower than that of control group (P<0.05).The difference of cell survival rate between lentivirus and control group was not significant (P>0.05).Based on the transfection efficiency and cell viability of this study,the lentiviral transfection method was the suitable way for transfection of PK15 cells,which provided a perfect method for high-efficiency transfection of PK15 cells in the future.