中文摘要：

目的利用慢病毒介导的RNA干扰技术构建人淋巴母细胞TK6细胞的PARP-1基因沉默细胞株,为后续PARP-1基因功能和表观遗传学调控机制的研究提供有效的工具细胞。方法利用瞬时转染方法筛选最佳干扰效果的shRNA片段,与慢病毒载体pLVX-shRNA1连接,形成重组质粒,DNA测序鉴定后进行病毒包装,转导TK6细胞,利用嘌呤霉素筛选得到稳定表达PARP-1siRNA的PARP-1沉默细胞株,并用定量PCR和Western blotting鉴定所构建的细胞株。结果转染shRNA1、shRNA2、shRNA3、shRNA4细胞的PARP-1相对表达量分别为0.289、0.538、0.375和0.474,故选择shRNA1作为干扰PARP-1基因最佳片段。用0.5μg/ml的嘌呤霉素成功筛选出PARP-1沉默细胞株和空载体对照细胞株,并从mRNA、蛋白质、细胞表型来检测PARP-1基因的干扰效率,抑制效率超过80%。结论成功构建PARP-1的siRNA慢病毒干扰载体,并筛选出PARP-1基因沉默的TK6细胞株,携带shRNA的慢病毒稳定、高效干扰PARP-1基因表达。

英文摘要：

Objective To silence PARP-1 gene in TK6 cells by lentiviral-mediated RNA interference technology and to provide an effective cell model for subsequent study of PARP-1 gene function and the mechanism of epigenetic regulation.Methods The best interference fragment was screened with transient transfection method, cloned into lentiviral vector, followed by DNA sequencing, virus packaging and then transduced to TK6 cells. Puromycin was used to select the defective cells which stably expressed PARP-1-shRNA. QPCR and Western blotting were used to identify the gene silencing efficiency. Results The relative expression of PARP-1 in four kinds of cells which transfected shRNA1, shRNA2, shRNA3, shRNA4 were 0.289,0.538, 0.375 and 0.474, respectively, so shRNA1 was the best interference fragment. PARP-1 silencing cell lines and empty vector control cells were successfully screened out with 0.5 μg/ml puromycin, and interference efficiency of PARP-1 gene was detected based on levels of mRNA, protein, cell phenotype, inhibition efficiency was more than 80%. Conclusion PARP-1siRNA lentiviral interference vector has been constructed successfully, which efficiently and stably interferes PARP-1 gene expression, and the PARP-1 gene-silenced TK6 cell line is screened out.