中文摘要：

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oligoDNA，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westernblot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westernblot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。

英文摘要：

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.