不同试剂转染RIP140-siRNA至库普弗细胞的效率及毒性比较

发布时间：2018-04-17 08:38

本文选题：肝脏库普弗细胞 + 转染效率　；参考：《南方医科大学学报》2015年12期

【摘要】：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(P0.05)。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。
[Abstract]:Objective to compare the transfection efficiency and cytotoxicity of different transfection reagents (RIP140-siRNA) into liver Kupffer cells, and to find out the best transfection methods and conditions of liver Kupffer cells.Methods Hepatic Kupffer cells were studied. RIP140-siRNA labeled with green fluorescent protein (GFP) was used as reporter gene. Lipofectamine 2000, X-treme GENE siRNA Transfection Reagentand lentivirus selected by purine mycin were used as transfection reagents.The effect of cell transfection was observed under fluorescence inverted microscope. The expression of RIP140 was analyzed by laser scanning confocal microscopy (LSCM) and apoptosis was detected by flow cytometry (FCM) and the inhibition of cell proliferation was detected by flow cytometry (FCM) and CCK-8 respectively.The RNA and protein were extracted from the cells, and the expression of genes and proteins after transfection of RIP140-siRNA was detected by RT-RCR and Western blot.Results for liver Kupffer cells, the transfection efficiency of lentivirus screened by purine mycin was the highest (over 90%), and that of lipofectamine 2000 was the worst.The results of flow cytometry and CCK-8 analysis showed that the cytotoxicity of Roche reagent was the least and the original morphology of the cells was observed, while the cytotoxicity of lentivirus was the highest in lipofectamine 2000, and most of the cells lost their original morphology.The cell cleavage state. RT-RCR and Western blot experiments showed that the expression of Lentivirus transfected RIP140-siRNA in Kupffer cell group was significantly lower than that in lipofectamine 2000 and Roche reagent transfected Kupffer cells group in both gene and protein level.Conclusion in the aspect of reagent transfection, lentivirus transfection can achieve ideal transfection efficiency and less cytotoxicity, and the conditional controllability and stability are better for the primary cell liver Kupffer cells.
【作者单位】：重庆医科大学第二临床学院;重庆医科大学附属第二医院肝胆外科;
【基金】：国家自然科学基金(81470899,81170442)~~
【分类号】：R575

【相似文献】