缺氧诱导因子-1α shRNA重组质粒的构建及对人结肠癌细胞生长的影响  

Construction of HIF-1 alpha gene specific short hairpin RNA expressing plasmid and effect on human colon carcinoma cell

作　　者：王忱[1] 史玉荣[1] 胡凡果[1] 牛瑞芳[1] 郝希山[1] 

机构地区：[1]天津医科大学附属肿瘤医院,天津300060

出　　处：《山东医药》2008年第26期18-20,共3页Shandong Medical Journal

摘　　要：目的构建靶向缺氧诱导因子-1α特异性短发卡RNA(shRNA)的真核表达载体,为结肠癌的靶向治疗奠定基础。方法设计、合成针对缺氧诱导因子-1α的特异性短链寡核苷酸,构建缺氧诱导因子-1α特异性shR-NA的重组质粒,稳定转染结肠癌SW 480细胞;采用氯化钴制备缺氧诱导培养基,模拟肿瘤缺氧状态。采用实时定量聚合酶链反应和免疫印迹法检测转染前后SW 480细胞中缺氧诱导因子-1α的表达;MTT法检测SW 480细胞活性。结果经酶切和测序鉴定,成功构建H IF-1α特异性shRNA的重组质粒,并能转染SW 480细胞。转染后,缺氧诱导因子-1αmRNA和蛋白水平表达分别下降约86.1%和79.7%,肿瘤细胞增殖活性明显降低。结论运用pGenesil-1质粒载体构建的靶向H IF-1α特异性shRNA的重组质粒已成功构建,该质粒可转染SW 480细胞,有效抑制H IF-1α的表达;本研究可为结肠癌的靶向治疗提供新方法。Objective To investigated the effect of short hairpin RNA （shRNA） targeting hypoxia inducible-1α （HIF-1α） on the human colon carcinoma SW480 cell line under hypoxia conditions. Methods The hypoxia environment was achieved by treating cells with CoCL2. The shRNA targeting HIF-1α was constructed and transfeeted into SW480 cells through Lipofectamine 2000. The mRNA and protein levels of HIF-1α were detected by real time PCR and western-blot. Cell poliferation was examined by MTT. Results The HIF-shRNA expression plasmid was successfully constructed and transfeeted into SW480 cells. The expression inhibition rate was 86.1% and 79.7% at mRNA level and protein level. The celluar proliferation of carcinoma also decreased. Conclusion A HIF-shRNA constructed by pGenesil-1 can be sucessfully transfected into SW480 cells,which can inhibit the expression of HIF-1α, supress the growth of SW480 cells.

关 键 词：结肠肿瘤 结肠癌 短发卡RNA 缺氧诱导因子-1Α 

分 类 号：R735.3[医药卫生—肿瘤]