E2F-1基因反转录病毒载体的构建及其对人胃癌细胞MGC-803增殖的影响  

Construction of retroviral expression vector of E2F-1 gene and its effect on proliferation of gastric cancer cell line MGC-803

作　　者：钱倩[1] 李雷[1] 王晓通[1] 苑汐子[1] 谢玉波[2] 肖强[1] 

机构地区：[1]广西医科大学第一附属医院胃肠腺体外科,南宁530021 [2]广西医科大学第一附属医院麻醉科,南宁530021

出　　处：《肿瘤》2010年第5期381-385,共5页Tumor

基　　金：国家自然科学基金资助项目(编号:30860273)

摘　　要：目的:通过构建人同源基因E2F-1反转录病毒表达载体,观察E2F-1过表达对胃癌细胞MGC-803增殖的影响。方法:应用基因重组技术将E2F-1基因克隆到反转录病毒载体pLXSN中,并将经酶切和RT-PCR检测正确的重组载体转染PA317包装细胞,收集病毒上清液感染胃癌细胞MGC-803。应用RT-PCR和Western印迹法检测MGC-803/pLXSN-E2F-1细胞中E2F-1 mRNA和蛋白的表达情况。应用克隆形成实验和MTT法检测细胞的增殖情况。结果:经酶切和RT-PCR检测结果显示,成功构建E2F-1反转录病毒表达载体。与MGC-803和MGC-803/pLXSN组相比,MGC-803/pLXSN-E2F-1组E2F-1mRNA和蛋白的表达明显上调(P<0.05)。MGC-803/pLXSN-E2F-1组的克隆形成率为48%,明显低于MGC-803组的81%和MGC-803/pLXSN组的77%,差异有统计学意义(P<0.01)。MGC-803/pLXSN-E2F-1组的细胞增殖率明显慢于MGC-803组和MGC-803/pLXSN组(P<0.01)。结论:成功构建了E2F-1基因过表达的反转录病毒表达载体,并将其成功感染MGC-803细胞,E2F-1过表达可抑制MGC-803细胞的增殖。Objective：To construct a novel retroviral expression vector of human homologous gene E2F-1 and observe the effect of E2F-1 gene over-expression on the proliferation of gastric cancer cells MGC-803.Methods：E2F-1 was cloned and inserted into the retroviral expression vector pLXSN using gene recombination technique.The recombinant retroviral expression vector pLXSN-E2F-1 was identified by restriction endonuclease digestion and RT-PCR.The recombinant vector and the empty vector were infected into the packaging cell line PA317.The virus supernatant was collected and used to infect gastric cancer cells MGC-803.Western boltting and RT-PCR were used to detect the expression of the mRNA and protein of E2F-1 gene,respectively.Colony forming assay and MTT assay were used to detect the proliferation.Results：Restriction endonuclease digestion and RT-PCR revealed that the retroviral expression vector of E2F-1 gene was successfully constructed.Compared with the MGC-803 group and MGC-803/pLXSN group,the expression of E2F-1 in MGC-803/pLXSN-E2F-1 group was significantly up-regulated（P〈0.05）.The colony forming rate of MGC-803/pLXSN-E2F-1 group was 48%,which was significantly lower than the MGC-803 group（81%） and MGC-803/pLXSN group（77%）（P〈0.01）.The proliferation speed of MGC-803/pLXSN-E2F-1 group was significantly slower than that of MGC-803 group and MGC-803/pLXSN group（P〈0.01）.Conclusion：The retroviral expression vector of E2F-1 was successfully constructed and transfected into MGC-803 cells.Over-expression of E2F-1 inhibited the proliferation of the MGC-803 cells.

关 键 词：胃肿瘤 反转录病毒科 基因表达 细胞增殖 基因 E2F-1 细胞 MGC-803 

分 类 号：R735.2[医药卫生—肿瘤]