论文部分内容阅读
目的:构建针对HER2/neu的RNA干涉载体,观察对HER2/neu表达抑制及抑制后对非小细胞肺癌生长的影响.方法:设计和合成长度为64nt的脱氧寡核苷酸链,退火互补成双链,克隆至pSUPER质粒载体,转化DH5α菌株,提取质粒,转染过表达HER2/neu的肺腺癌细胞系SPCA1,RTPCR检测HER2/neu的mRNA表达,Westernblot和间接免疫荧光法检测HER2/neu的蛋白表达,FCM分析细胞周期变化,MTT法观察细胞生长.结果:肺腺癌细胞系SPCA1存在HER2/neu过表达;成功构建了HER2/neu特异性RNA干涉载体pSUPERsiHER2;瞬时转染SPCA1细胞,HER2/neu的mRNA及蛋白表达均降低;G1期细胞较亲代增加9.9%;肿瘤细胞增殖速度减慢(P<0.05).结论:成功构建了HER2/neuRNA干涉载体,转染后可有效降低肺腺癌细胞系SPCA1HER2/neu的mRNA转录及蛋白水平表达,阻滞转染细胞于G1期,造成转染细胞生长速度减慢,这有望为肺癌基因治疗提供一种选择手段.
OBJECTIVE: To construct an RNA interference vector targeting HER2/neu and to observe the effect of HER2/neu inhibition and its inhibition on the growth of non-small cell lung cancer. Methods: Design and synthesis of a deoxy-oligonucleotide chain with a length of 64 nt and annealing to complement each other. Double-stranded, cloned into pSUPER plasmid vector, transformed DH5α strain, extracted plasmid, transfected overexpressed HER2/neu lung adenocarcinoma cell line SPCA1, RTPCR detected HER2/neu mRNA expression, Western blot and indirect immunofluorescence detected HER2/neu Protein expression, FCM analysis of cell cycle changes, MTT assay to observe cell growth. Results: Overexpression of HER2/neu in lung adenocarcinoma cell line SPCA1; successful construction of HER2/neu specific RNA interference vector pSUPERsiHER2; transient transfection of SPCA1 cells, The mRNA and protein expression of HER2/neu was decreased; the G1 phase cells were increased by 9.9% compared with the parental group; the proliferation rate of tumor cells was slowed down (P<0.05). Conclusion: The HER2/neuRNA interference vector was successfully constructed, and the lungs can be effectively reduced after transfection. The mRNA and protein expression of SPCA1HER2/neu in the adenocarcinoma cell line blocked the transfected cells in the G1 phase, resulting in slower growth of the transfected cells, which is expected to provide a selection method for gene therapy of lung cancer.