Sirt1基因shRNA干扰载体构建及其对细胞增殖和凋亡的影响

Construction of Sirt1 shRNA Interfering Vector and Its Effects on Cell Proliferation and Apoptosis

本实验目的在于构建Sirt1 shRNA干扰载体,探讨Sirt1对HepG2、A549和293T细胞增殖和凋亡的影响。设计并合成Sirt1的shRNA序列,重组到pGenesil-1.0质粒载体中筛选出pGenesil-1.0-Sirt1阳性克隆并测序。将构建好载体转染HepG2、A549和293T细胞,用RT-PCR、Western blot检测各细胞中Sirt1表达水平,MTT测细胞增殖活性,加入阿霉素处理三组细胞后,用MTT检测细胞凋亡情况。结果表明成功构建Sirt1 shRNA干扰载体,各细胞株中Sirt1表达水平明显下降,Sirt1 shRNA克隆细胞株增殖能力降低,阿霉素处理后,各细胞株抗凋亡能力明显减弱,Sirt1在维持细胞增殖及抗细胞DNA损伤凋亡方面发挥重要作用,为进一步研究提供理论依据。

This study was aimed to construct Sirtl shRNA interfering vector and to analyze the effects of Sirtl on cell proliferation and apoptosis in HepG2, A549 and 293T cell lines. To design and synthisize Sift1 shRNA sequence then recombinate it to pGenesil-1.0 plasmid, the positive pGenesil-1.0-Sift1 vector clone was screened by effective detections and sequencing. The vectors were transfected into HepG2, A549, 293T cell lines, and Sirtl expression levels in these clones were detected by RT-PCR and Western-blot. These clone cell proliferation activities were detec- ted by MTT, and these cells apoptosis incidences were detected by MTT experiment after treated with DOX. The re- sults showed that Sirtl shRNA interfering vectors were successfully screened. The levels of Sirtl expression in HepG2-sh, A549-sh and 293T-sh cells were significantly reduced compared with their control cells. It was indicated that the proliferation activities of these cells were impaired and anti-apoptosis capabilities of HepG2-sh, A549-sh and 293T-sh were also impaired notably. Sift1 took an important role in maintaining cell proliferation and resisting cell apoptosis caused by DNA damage, and this result also provided theoretical information for the further research.