肠上皮细胞RNA干扰Hsp70基因转染条件的研究

Research on Transfection Condition of RNA Interference Targeting Hsp70 Gene in Intestinal Epithelial Cells

旨在研究肠上皮细胞(IEC-6)RNA干扰效率的转染条件,并对4对Hsp70干扰序列进行筛选,为进一步以RNA干扰(RNAi)技术研究Hsp70对胃肠道的保护功能以及介导信号通路的机理打下基础。以24孔培养板培养IEC-6细胞,利用阳离子脂质体转染FAM-siRNA片段进入IEC-6细胞,采用荧光倒置显微镜和流式细胞仪检测FAM-siRNA和转染试剂Lipo-fectamine 2000不同剂量比例对转染效率的影响,并进一步采用RT-PCR和Western blotting方法对Hsp70干扰序列进行筛选。试验结果表明,采用脂质体转染法可获得较高的转染效率,其中以80 nmol/L FAM-siRNA+1μL Lipofectamine 2000组的转染效率最高(85.77%)。针对4个不同靶位点,进行Hsp70基因干涉,结果表明,4个组中Hsp70 mRNA的表达显著下降,其中ol-igo 4组的基因表达极显著降低。Western blotting的结果表明,oligo 2、oligo 3和oligo 4组中的Hsp70蛋白表达极显著降低。最终确定80 nmol/L FAM-siRNA+1μL Lipofectamine 2000为最佳转染条件,以oligo 4(Hsp70的基因位点为3672-3692)为最佳RNA干扰靶基因。

This study was conducted to investigate the transfection condition for affecting RNA interference efficiency on intestinal epithelial cells（IEC-6）cells,and determine the highest efficiency of RNA interference targeting Hsp70,therefore,it could lay a foundation for the further research on the protection of Hsp70 in gastro-intestinal tract and cell signal transduction of Hsp70 by the method of RNA interference.IEC-6 cells were cultured in 24-well plate.With the help of cation liposome,the transfection of FAM-siRNA fragment into IEC-6 was carried out,and the effect of FAM-siRNA and Lipofectamine 2000 in different proportions on the transfection efficiency was examined with fluorescence microscope and flow cytometry.Expression of Hsp70 was determined by RT-PCR and Western blotting.The results showed that transfect efficiency was the highest（85.77%）in the group of 80 nmol/L FAM-siRNA ＋ 1 μL Lipofectamine 2000.The results of transfection of a small interfering RNA targeting Hsp70 showed that the expression of Hsp70 mRNA were significantly decreased in four groups and expression of Hsp70 proteins were significantly decreased in oligo 2,oligo 3 and oligo 4 groups.In conclusion,under the transfection condition of 80 nmol/L FAM-siRNA ＋ 1 μL Lipofectamine 2000,IEC-6 has the highest transfection efficiency during RNA interference and oligo 4（Target site：3 672-3 692）has the highest interference efficiency targeting Hsp70 gene.