E2F1的siRNA真核表达载体转染对胆管癌细胞的作用

目的:探讨E2F1蛋白信号的转导通路对胆管癌细胞凋亡的影响中的作用。方法:根据E2F1基因cDNA序列，设计针对目的基因的4个siRNA靶序列，将其插入U6启动子下游，克隆到真核表达载体pGsensil中，通过酶切鉴定和DNA测序鉴定。采用脂质体介导法将构建好的4个siRNA表达载体分别转染于QBC939细胞中；以RT-PCR检测E2F1基因在转染前后的表达，流式细胞术检测转染后对细胞凋亡率的影响。结果:重组质粒酶切后呈线性化，酶切鉴定及DNA测序结果显示插入片断正确。转染48h后，4个siRNA表达载体均抑制了E2F1 mRNA的表达，沉默E2F1基因后细胞的凋亡率显著增加。结论:E2F1的siRNA真核表达载体转染QBC939细胞后，该表达载体具有抑制E2F1基因表达、促进细胞凋亡的功能，可以用于后续胆管癌的实验研究。

The effect of siRNA eukaryotic expression vector transfection of E2F1 on cholangiocarcinoma cells

Abstract:Objective:To study the action of E2F1 protein signal transmission pathway to influence the apoptosis of cholangiocarcinoma QBC939 cells.Methods :Four siRNAs were designed according to the coding sequence of E2F1 gene, and cloned into the downstream of U6 promoter of pGenesil. The constructed recombinant was analyzed and identified by AseⅠ endonuclease digestion and DNA sequencing. The siRNA constructs were transfected into QBC939 cells via lipofectamine 2000. RT-PCR was used to measure the E2F1 mRNA expression in QBC939, and the rate of apoptosis of QBC939 was detected by the method of flow cytometry after transfection.Results:The constructed psiRNA plasmid digested with AseⅠwas linearized. The sequencing result confirmed that the sequence of inserted fragment was correct. The expression of E2F1 mRNA was greatly inhibited at 48h after transfection, and the rate of apoptosis of QBC939 increased significantly after transfection.Conclusions:Eukaryotic expression vector of siRNA targeting E2F1 gene can specifically inhibit E2F1 expression and promote the apoptosis of QBC939 cell. It can be used for later experimental research in cholangiocarcinoma.