X线修复交叉互补基因1缺陷细胞株的建立及其蛋白的表达

Establishment of XRCC1-deficient cell and its cell biological characteristics

目的运用载体介导的RNA干扰技术靶向抑制人X线修复交叉互补基因1(XRCC1)在支气管上皮细胞中的表达,为研究人XRCC1蛋白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XRCC1 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XRCC1蛋白表达情况。结果在转染重组子的细胞中,XRCC1蛋白的表达明显下调,仅相当于正常细胞的38.2%。结论人支气管上皮细胞人X线修复交叉互补基因1的靶向抑制成功。

Objective To establish XRCCl-defecient cell line through RNA interference, and investigate its cell biological characteristic, and thus lay a basis for the further study on the function of XRCC1.Methods HBE cell was transfected with siRNA Eukaryotic Expression Vector for XRCC1 to establish XRCCl-defecient cell strain. After G418 resistant selection and the successful transfection was identified by fluorescence microscope, the protein expression levels of XRCC1 gene in HBE and HBEX were detected by western blotting so as to verify the effect of interference. Results Successful transfection was confirmed by the cells capable of emitting green light under fluorescent microscope. Western blotting showed that the protein expression level of XRCC1 gene in HBEX was 38.2% of that in HBE. Conclusion The RNA interference of XRCC1 gene in human bronchial epithelial cells was successful.