Rheb和Rheb＇D60K突变基因重组慢病毒载体的构建、鉴定及其对肝癌细胞凋亡的影响  

Construction of recombinant lentiviral vectors containing Rheb gene and its mutant Rheb'D60K gene and their expression in human liver cancer cells

作　　者：陈可和[1] 梁博[2] 邹镇洪[2] 韩泽龙[2] 潘金飞[1] 刘安玲[2] 

机构地区：[1]广西壮族自治区人民医院临床肿瘤中心,广西南宁530021 [2]南方医科大学基础医学院细胞生物学教研室,广东广州510515

出　　处：《南方医科大学学报》2012年第3期341-344,348,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(30900555)~~

摘　　要：目的构建携带Rheb和Rheb'D60K突变基因的重组慢病毒载体,并鉴定其在MCF-7细胞中的表达效果,观察其对肝癌细胞凋亡的影响。方法 PCR法扩增Rheb和Rheb'D60K突变基因,构建重组质粒LV31-Rheb-WT、LV31-Rheb-D60K,脂质体法将重组慢病毒载体和包装质粒混合物共转染HEK-293 FT细胞,包装产生慢病毒颗粒。将病毒感染MCF-7细胞后Western blotting检测Rheb及PS6蛋白的表达;将病毒感染SK-HEP-1细胞饥饿处理后观察其凋亡情况。结果 PCR及测序表明成功构建了LV31-Rheb-WT、LV31-Rheb-D60K重组慢病毒载体。Western blotting结果显示慢病毒LV31-Rheb-WT感染的MCF-7细胞内Rheb下游的PS6蛋白表达多于慢病毒LV31-Rheb-D60K感染的MCF-7细胞。显微镜下慢病毒LV31-Rheb-D60K感染的SK-HEP-1细胞饥饿后凋亡数目多于慢病毒LV31-Rheb-WT感染的SK-HEP-1细胞。结论本研究成功构建了Rheb和Rheb'D60K突变基因重组慢病毒载体,初步观察到该突变基因重组慢病毒载体可诱导肝癌细胞凋亡,为进一步研究Rheb基因在肝癌发生发展中的作用,进而开展肝癌的临床靶向治疗研究奠定了基础。Objective To construct recombinant lentiviral vectors carrying Rheb gene and its mutant Rheb＇D60K gene,and examine their expression in human liver cancer cells.Methods Rheb gene was amplified by PCR to construct the recombinant plasmid LV31-Rheb-WT and LV31-Rheb-D60K.HEK-293 FT cells were contransfected with the recombinant lentiviral vector together with a lentiviral package plasmid to produce the lentiviral particles.The expression of PS6 protein was detected in the lentivirus-infected MCF-7 cells.The apoptosis of SK-HEP-1 cells transfected with LV31-Rheb-WT or LV31-Rheb-D60K was observed.Results The recombinant LV31-Rheb-WT and LV31-Rheb-D60K vectors were confirmed by PCR and DNA sequencing.Western blotting showed that PS6 protein expression was increased in LV31-Rheb-WT-transfected cells while decreased in LV31-Rheb-D60K-transfected cells.LV31-Rheb-D60K-transfected SK-HEP-1 cells showed more obvious apoptosis after starvation than LV31-Rheb-WT-transfected cells.Conclusion Lentiviral vectors carrying Rheb gene and its mutant has been successfully constructed,which can be useful in further investigation of the role of Rheb gene in cancer cells.

关 键 词：RHEB 慢病毒载体 MCF-7细胞株 乳腺癌 SK-HEP-1细胞株 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]