Smad2特异性siRNA转染肝星状细胞的效率检测

Detection of Smad2 targeted siRNA transfection efficiency in hepatic stellate cells

关键词:  RNA干扰  肝星状细胞  肝纤维化  基因转染

Key words:  RNA interference (RNAi)  hepatic stellate cytes（HSCs）  liver fibrosis  gene transfection

摘要:

［目的］提高siRNA表达载体在肝星状细胞（hepatic stellate cells,HSCs）中的转染效率。［方法］pEGFP-C1-CR4转染原代HSCs、HSC－T6细胞株，实验根据质粒DNA和阳离子脂质体的不同比例分为4组: 1 μg/0 μL、1 μg/1 μL、1 μg/2 μL、1 μg/4 μL，1μg/0 μL为对照组，其余3组为实验组。转染48 h后，使用荧光显微镜观察转染情况，同时流式细胞仪计算转染效率。［结果］ 质粒DNA和阳离子脂质体的比例为1 μg/2 μL和1 μg/4 μL时，原代HSCs转染效率为（56.55±3.12）%和（58.62±2.03）%（P>0.05），HSC－T6细胞株转染效率为（24.52±3.55）％和（25.49±2.33）％（P>0.05）。同样比例条件下，原代HSCs的转染效率显著高于HSC－T6细胞株的转染效率（P<0.05）。在实验组质粒、脂质体比例为1 μg/2 μL和1 μg/4 μL时，同种细胞的转染效率均无显著差别（P>0.05）。［结论］采用质粒DNA和阳离子脂质体的比例为1 μg/2μL、1 μg/4 μL的条件，均能够在HSCs和HSC－T6中获得最佳转染效率，但前者更为经济。

Abstract:

［Objective］ To increase the transfection efficiency of siRNA expression constructs transfection in hepatic stellate cells in vitro. ［Methods］ HSCs and HSC-T6 cell line were transfected with pEGFP-C1-CR4 plasmid DNA. According to the different ratio of plasmid DNA/Lipofectamine 2000 (Life technologies), the transfection protocols were divided into 4 groups: naked DNA, 1 μg/1 μL, 1 μg/2 μL and 1 μg/4 μL. 48 hours later, transfection efficiencies of different protocols were evaluated by fluorescent microscopy observation and flow cytometer counting. ［Results］ The transfection efficiencies in 1 μg/2 μL and 1 μg/4 μL groups were （56.55±3.12）% and（58.62±2.03）% in primary HSCs（P>0.05）, （24.52±3.55）％ and（25.49±2.33）％ in HSC－T6 cell line（P>0.05）. The efficencies were significant higher in HSCs than those in HSC-T6 cell line （P<0.05）. There was no significant difference on each kind of cell between the 1 μg/2 μL and 1μg/4μL groups in efficacy（P>0.05）. ［Conclusion］ In our study, 1 μg/2 μL (DNA /LIPO) is the most economical and optimal ratio to get best transfection efficiency in HSCs and HSC-T6 cell line.