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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 establish an efficient and stable poly-ADP-ribose hydrolase (PARG) deficient cell line of human bronchial epithelial cells (16HBE) in order to study the role of PARG in environmental toxicant-induced cell injury and to understand poly-ADP-ribosylation Laid the foundation for the function. Methods The oligo DNA encoding a short hairpin RNA (shRNA) targeting human PARG gene was designed and synthesized. The oligo DNA was inserted into the eukaryotic expression vector plko.1-puro to construct a recombinant plasmid. After successful identification, the recombinant plasmid was transfected into normal 16HBE cells, the expression of PARG gene and protein of the selected cell lines was detected by RT-PCR and western blot, and the growth cycle of the constructed cell lines was analyzed by flow cytometry. Results The recombinant plasmids were identified by sequencing. RT-PCR and western blot showed that the interference efficiency of the selected cell lines was over 80% with significant effect (compared with normal cells, P <0.05); and the growth cycle of defective cell lines No significant change (P> 0.05). Conclusion The study successfully constructed a highly efficient and stable PARG-deficient cell line of human bronchial epithelial cells and provided a reliable platform for clarifying the mechanism of ADP-ribosylation reaction and its biological effects.