不同试剂转染RIP140-siRNA至库普弗细胞的效率及毒性比较

Comparison of efficiency and cytotoxicity of different transfection reagents in transfecting RIP140-siRNA into Kupffer cells

目的对比不同的转染试剂转染RIP140-siRNA至肝脏库普弗细胞的转染效率及它们对肝脏库普弗细胞的细胞毒性,从而寻找出最佳的肝脏库普弗细胞试剂转染方法和条件。方法以肝脏库普弗细胞为研究对象,以绿色荧光蛋白(GFP)标记的RIP140-siRNA为报告基因(reporter gene),采用lipofectamine 2000,罗氏试剂(X-treme GENE siRNA Transfection Reagent)及嘌呤霉素筛选的慢病毒(1.0×108TU/m L)作为转染试剂,荧光倒置显微镜下观察细胞转染效果,激光扫描共聚焦显微镜分析不同试剂转染后细胞RIP140的表达,流式细胞术检测各组细胞凋亡,CCK-8检测各组细胞增殖抑制情况。收集细胞并进行裂解,提取细胞RNA与蛋白质,运用RT-RCR和Western blot实验法检测转染RIP140-siRNA后的基因及蛋白质表达情况。结果对于肝脏库普弗细胞而言,在转染效率方面:嘌呤霉素筛选的慢病毒转染效率最高,可达90%以上;罗氏试剂其次;lipofectamine2000效果最差。在试剂的细胞毒性方面:流式细胞术及CCK-8检测结果显示罗氏试剂的细胞毒性最小,细胞可见其原有形态;慢病毒其次;lipofectamine 2000细胞毒性最大,可见多数细胞失去原有形态,并存在细胞裂解状态。RT-RCR和Western blot实验显示慢病毒转染RIP140-siRNA的肝脏库普氏细胞组无论在基因方面还是在蛋白质水平均明显低表达于lipofectamine 2000和罗氏试剂所转染的肝脏库普弗细胞组(P<0.05)。结论对于原代细胞肝脏库普弗细胞,在试剂转染方面,慢病毒转染方法可以达到理想的转染效率和较小的细胞毒性,且条件可控性与稳定性方面更加优越。

Objective To compare the efficiency and cytotoxicity of different transfection reagents used in transfection of RIP140-siRNA into Kupffer cells to optimize the transfection conditions. Methods Kupffer cells were transfected with RIP140- siRNA labeled with GFP as the reporter gene using lipofectamine 2000, Roche reagent（X- treme GENE siRNA Transfection Reagent）and puro screening lentivirus（1.0 × 108TU/m L） as the transfection reagents. The transfection effect was observed under a fluorescent inverted microscope, and laser scanning confocal microscopy was used to analyze RIP140 expression in trasnfected Kupffer cells. Flow cytometry was performed to detect cell apoptosis, and CCK- 8 test was used to evaluate the cell proliferation inhibition. RT-RCR and Western blotting were performed to detect the expressions of RIP140 m RNA and protein in the trasnfected cells. Results Puro screening lentivirus yielded the highest cell transfection efficiency, which exceeded 90%,followed by Roche reagent and then by lipofectamine 2000. Flow cytometry and CCK-8 test showed that the cytotoxicity was the mildest with Roche reagent, moderate with lentivirus, and severe with lipofectamine 2000. The cells trasnfected with lentivirus showed a significantly lower RIP140 expression than cells trasnfected with lipofectamine 2000 and Roche reagent（P〈0.05）. Conclusion In Kupffer cells, lentivirus-mediated transfection, as compared with the other two trasnfection reagents, can achieve good transfection efficiency with a relativelty low cytotoxicity, and allows for better controllability and stability of the trasnfectiion conditions.