慢病毒介导的mTOR敲低肝癌细胞株HepG2的构建及初步功能检测  

Construction and Preliminary Function Assay of a Lentivirus-Medi ated mTOR Knock-Down Hepatoma Cell Line HepG2

作　　者：刘家宏[1,2] 徐小洁[1] 符静[3] 范忠义[1] 吕朝晖[3] 陆菊明[3] 肖文华[2] 朱建华[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院第一附属医院肿瘤一科,北京100037 [3]解放军总医院内分泌科,北京100853

出　　处：《生物技术通讯》2013年第4期467-470,共4页Letters in Biotechnology

基　　金：国家自然科学基金(30872939;31100604)

摘　　要：目的:建立高效稳定的哺乳动物雷帕霉素靶蛋白(mTOR)小干扰RNA(siRNA)细胞导入方法,并对mTOR敲低的HepG2肝癌细胞株的功能进行初步检测。方法:构建了2条不同的人mTOR慢病毒siRNA载体pLenti-H1/mTOR siRNA,与3个包装质粒共转染293T细胞,包装成慢病毒后感染HepG2细胞;经嘌呤霉素筛选2周后,收集细胞进行Western印迹,检测mTOR敲减效果及其下游基因c-myc、周期蛋白D1(cyclinD1)表达水平及4E-BP1、S6K1磷酸化水平的变化。结果:RT-PCR和Western印迹结果显示,构建的pLenti-H1/mTOR siRNA能有效抑制mTOR基因的表达,敲低了mTOR蛋白水平,且沉默mTOR后其下游基因c-myc、CyclinD1的表达水平及4E-BP1、S6K1磷酸化水平降低。结论:构建了慢病毒介导RNA干扰mTOR表达载体,为进一步研究mTOR通路奠定了实验基础。Objective： To establish an efficient and stable method for mammalian target of rapamycin（mTOR） small interfering RNA（siRNA） delivery into hepatoma cell line HepG2 and assay its function preliminarily. Meth-ods： Two different pLenti-H1/mTOR siRNA were constructed, and they were transfected into 293T cells together with three viral packaging vectors to produce lentivirus particles. After HepG2 cells were infected with the harvest- ed viruses and experienced screening for 2 weeks with puromycin, they were collected to examine expression and function of roTOR by Western blot and quantitive RT-PCR（qPCR） analysis. Results： Western blot and qPCR showed that pLenti-H1/mTOR siRNA could suppress the roTOR gene expression. Suppression of mTOR could mark-edly down-regulate the phosphorylation level of its downstream targets such as 4E-BP1 and S6K1, and expression level of c-myc and cyclinD! genes. Conclusion： The lentivirus-mediated mTOR siRNA were obtained, which lays the foundation for further research on mTOR signaling pathway.

关 键 词：哺乳动物雷帕霉素靶蛋白 RNA干扰 慢病毒 

分 类 号：Q78[生物学—分子生物学]