人支气管上皮细胞聚-ADP-核糖水解酶缺陷细胞株的构建及鉴定  

Construction and identification of human bronchial epithelial cells of Poly(ADP-ribose) glycohydrolase deficient cell strain

作　　者：蔡剑锋[1] 黄海燕[2] 刘建军[2] 杨淋清[2] 吴德生[2] 夏菠[2] 毛吉炎[2] 胡恭华[2] 刘庆成[2] 李习艺[1] 庄志雄[2] 

机构地区：[1]广西医科大学公共卫生学院,广西南宁530021 [2]深圳市疾病预防控制中心毒理研究室

出　　处：《毒理学杂志》2012年第6期415-419,共5页Journal of Toxicology

基　　金：国家自然科学基金项目(81001261);深圳市科技计划项目(201002101);深圳市重点实验室提升项目(CXB201005260068A)

摘　　要：目的建立高效、稳定的人支气管上皮细胞(16HBE)的聚-ADP-核糖水解酶(PARG)缺陷细胞株,为研究PARG在环境毒物诱导细胞损伤中的作用及了解聚-ADP-核糖基化反应的功能奠定基础。方法设计并合成能编码特异靶向人PARG基因的短发夹RNA(shRNA)的oligo DNA,将之插入到真核表达载体plko.1-puro中构建重组质粒,鉴定成功后转染至正常的16HBE细胞中,RT-PCR及western blot检测完成筛选细胞株的PARG基因及蛋白水平的表达,并使用流式细胞仪分析构建成功细胞株的生长周期变化。结果重组质粒测序鉴定正确;RT-PCR及western blot检测显示最终选定的缺陷细胞株干扰效率达80%以上,效果显著(与正常细胞相比,P<0.05);且缺陷细胞株的生长周期未见明显变化(P>0.05)。结论该研究成功构建了高效、稳定的人支气管上皮细胞的PARG缺陷细胞株,为阐明ADP-核糖基化反应的作用机制及其生物学效应提供了可靠的平台。Objective To gain insight into the function of PARG and Poly （ADP-ribosyl）ation in response to stress, we used lentiviral gene silencing to generate 16HBE cell lines with stably suppressed PARG. Methods A pair of complementary small hairpin RNA （shRNA） oligonucleotides targeting the PARG gene were designed, sythesized, annealed and inserted into plko. 1-puro vector. After transfecting to normal 16HBE ceils, PARG was quantified in transfected cells at both protein and mRNA levels, and flow cytometry analysis was performed to analysis cell cycle progression. Results The recombinant plasmid DNA sequence was correct; RT-PCR and western blot detection both displayed that the interference efficiency of defective cell line could be more than 80% （ P 〈 O. 05 ） ; Compared with the control, the cell cycle of both shPARG cells and shRNAc cells was not significantly different （P 〉 0.05 ）. Conclusion A cellular model deficient in all PARG is successfully constructed and can be used for further study the role of the PARG and Poly（ADP-ribosyl） ation.

关 键 词：聚-ADP-核糖基化 聚-ADP-核糖水解酶(PARG) RNA干扰 慢病毒载体 人支气管上皮细胞(16HBE细胞) 

分 类 号：R99[医药卫生—毒理学]