【作者】 胡大林； 庄志雄； 伍德娥； 何云； 纪卫东； 袁建辉； 方道奎； 沙焱； 杨建平； 涂晓志；

【Author】 HU Da-lin,ZHUANG Zhi-xiong,HE Yun,JI Wei-dong,FANG Daokui, SHA Yan,TU Xiao-zhi,YANG Jian-ping,HU Gong-hua School of Public Health,SunYat-sen University,Guangzhou 510080,China

【机构】 佛山科学技术学院医学院； 中山大学公共卫生学院预防医学系； 深圳市疾病预防控制中心；

【摘要】 目的运用载体介导的RNA干扰技术靶向抑制人DNA聚合酶β基因在支气管上皮细胞中的表达,为研究DNA聚合酶β在环境化学污染物所致DNA损伤修复中的功能和机制作准备。方法利用分子克隆技术构建含pU6启动子的人DNA聚合酶β基因RNA干扰特异性绿色荧光蛋白C1载体重组子"pEGFP-C1-U6-dsRNA";以脂质体法将载体重组子转染人支气管上皮细胞,同时以空白细胞和空载体转染细胞作对照;在经G418筛选后,以荧光显微成像技术观察细胞的转染效果,以蛋白印迹法分析转染后细胞中的DNA聚合酶β表达情况。结果在转染重组子的细胞中,DNA聚合酶β的表达明显下调,仅相当于正常细胞的17.3%。结论人支气管上皮细胞DNA聚合酶β基因的靶向抑制成功。更多还原

【Abstract】 To knock down the expression of polymerase beta gene in human bronchial epithelial cells with technology ofvector-mediated RNA interference(RNAi),to provide research tool for the study on the functions and mechanisms of polymerase beta in repairing of DNA damaged by environmental chemical pollutants(ECPs).Methods Technology of molecular clone was used to construct the recombination vector of"pEGFP-C1-U6-dsRNA"for the polymerase beta RNAi.The recombinants were transfected into human bronchial epithelial cells with kit of liperfectamine 2000.The control groups included normal human bronchial epithelial cells and human bronchial epithelial cells transfected with"pEGFP-C1".Cells were screened by G418,then technology of fluorescence microscopy imaging was used to observe the result of transrfection.The expression level of polymerase beta was detected by Western blotting.Results The expression level of polymerase beta in human bronchial epithelial cells transfected with the recombinant of"pEGFP-C1-U6-dsRNA"was about 17.3%of what in the normal cells.Conclusion The RNAi of polymerase beta gene in human bronchial epithelial cells was successful.更多还原