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目的建立并鉴定聚ADP核糖聚合酶1(hPARP1)缺陷细胞株,用于研究hPARP1基因的作用机制及其缺陷与DNA损伤发生的关系。方法将用已经构建的pEGFPC1shRNA(包含两个PARP1基因及一个阴性对照)转染支气管上皮细胞(16HBE),使之在16HBE中表达,转染后命名为16HBEP1、16HBEP2及16HBEN。用蛋白免疫印迹法鉴定转染细胞中hPARP1基因的表达水平。结果pEGFPC1shRNA在真核细胞成功表达;16HBEP1和16HBEP2的蛋白表达水平分别下降了84.3%及63.7%。结论hPARP1缺陷细胞株的成功建立和鉴定为hPARP1基因功能研究提供了一种有效手段。
OBJECTIVE: To establish and identify hPAP1-deficient cell lines and to investigate the mechanism of hPARP1 and its relationship with DNA damage. Methods Transfected bronchial epithelial cells (16HBE) with the constructed pEGFPC1 shRNA (containing two PARP1 genes and one negative control) were expressed in 16HBE. After transfection, they were named 16HBEP1, 16HBEP2 and 16HBEN. Western blotting was used to identify the expression level of hPARP1 gene in transfected cells. Results pEGFPC1 shRNA was successfully expressed in eukaryotic cells. The protein expression levels of 16HBEP1 and 16HBEP2 decreased by 84.3% and 63.7%, respectively. Conclusion The successful establishment and identification of hPARP1-deficient cell lines provide an effective means for the study of hPARP1 gene function.