靶向于EpCAM干扰载体的构建及其对结直肠癌细胞的影响周逢强1.doc

靶向于EpCAM干扰载体的构建及其对结直肠癌细胞的影响 周逢强1 齐艳美2 徐宏1 郭辉光 1 刘磊1 （1 滨州市人民医院普外科，2 滨州市人民医院消化内科） 摘要：目的：构建靶向作用于EpCAM的载体，探讨EpCam干扰后对结直肠癌细胞的影响。方法：选取人结直肠癌HT-29及HCT-166细胞株，将双链的miRNA oligo插入到miRNA表达载体pcDNATM6.2-GW/ EmGFPmiR中，使用LipofectamineTM2000分别对空质粒组(转染pcDNATM6.2-GW/ EmGFPmiR-neg)和干扰组(转染pcDNATM6.2-GW/ EmGFPmiR-EPCA M-1)进行质粒转染，无转染组为空白对照组。采用RT-PCR转染效率，Western检测Ep-CAM蛋白的表达，并采用MTT法检测各组细胞的增殖能力。结果：RT-PCR证实干扰组Ep-CAM表达量显著低于空质粒组和对照组（P0.001），Western检测显示干扰组Ep-CAM蛋白表达显著低于空质粒组和对照组，MTT法显示相对于空质粒组和对照组，干扰组细胞增殖能力显著受到抑制，差异具有统计学意义（P0.001）。结论：沉默Ep-CAM可显著的抑制结直肠癌细胞的增殖。 关键词：上皮生长因子；结直肠癌；载体构建 Construction of interference vector targeting EpCAM gene and effects of its expression on colorectal cancer cells Zhou feng –qiang,Qi Yan Mei, Guo hui Guang,Xu hong,Zhang Ze dong (1 Binzhou City Peoples Hospital General Surgery, 2 Peoples Hospital of Binzhou Gastroenterology) Abstract Objective: To construct an interference vector targeting the EpCAM gene, and explore its effects on colorectal cancer cells. Methods: HT-29 and HCT-166 colorectal cancer cell lines were selected as cell models in the present study. The double-stranded miRNA oligo were inserted into pcDNATM6.2-GW/EmGFPmiR, which is an expression vector of miRNA. LipofectamineTM2000 were used to transfer plasmid into the empty plasmid group (transfected pcDNATM6.2-GW/EmGFPmiR-neg) and the interference group (transfected pcDNATM6.2-GW/EmGFPmiR-EPCA M-1), and the non-transferred one was difinitioned control group. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the transfection efficiency. Western blot was used to detect Ep-CAM protein expression, and the cell proliferations in each group were detected by MTT. Results: RT-PCR confirmed that the Ep-CAM expression interference was significantly lower in the interference group than that of the empty plasmid group and the control group (P 0.01). Western analysis showed that Ep-CAM protein expression was significantly lower in interference group than that of the empty plasmid group a