E2F-1基因真核表达载体的构建及其对胃癌细胞株MKN-45增殖的影响  

Construction of a Eukaryotic Vector Expressing E2F-1 Gene and Its Effect on Proliferation of Gastric Cancer Cell Line MKN-45

作　　者：王长青[1] 肖强[1] 李雷[1] 谢玉波[2] 谭宁[1] 宫志伟[1] 

机构地区：[1]广西医科大学第一附属医院胃肠腺体外科,广西南宁530027 [2]广西医科大学第一附属医院麻醉科,广西南宁530027

出　　处：《癌症》2007年第10期1064-1068,共5页Chinese Journal of Cancer

基　　金：广西自然科学基金资助项目(No0640085)~~

摘　　要：背景与目的:E2F-1基因在多种肿瘤细胞中过表达,并表现出抑制或促进肿瘤细胞增殖的不同特性。本研究通过构建E2F-1基因真核表达载体,分析E2F-1过表达对胃腺癌细胞株MKN-45的影响。方法:巢式聚合酶链反应(polymerase chain reaction,PCR)扩增E2F-1基因片段,用pCMV-HA载体构建真核重组质粒,转染MKN-45细胞(转染COS-7细胞用以鉴定所构建的载体)。Western blot检测转染情况,并通过克隆形成实验、MTS法检测细胞增殖情况,流式细胞仪分析细胞周期,观察E2F-1过表达对MKN-45细胞的影响。结果:pCMV-E2F1-HA重组质粒成功转染MKN-45细胞。转染pCMV-E2F1-HA的MKN-45细胞形成的克隆数为4.7±1.8,明显少于转染空载体组的30.2±6.7(P<0.01)。转染pCMV-E2F1-HA的MKN-45细胞增殖随时间而减慢,第5、6、7天的细胞增殖率分别为73.5%、63.5%和56.1%,明显低于转染空载体组(P<0.01)。与转染空载体组相比,表达E2F-1基因的MKN-45细胞在G0/G1期所占比例明显提高(71.4%vs.59.7%,P<0.05),S期所占比例明显下降(18.7%vs.30.7%,P<0.05)。结论:成功构建E2F-1基因真核表达载体,E2F-1过表达可抑制MKN-45细胞的增殖。　BACKGROUND ＆ OBJECTIVE： E2F-1 is overexpressed in many tumors and functions as an oncogene or tumor suppressor gene. This study was to construct the recombinant eukaryotic vector expressing E2F-1 cDNA, and explore the effect of E2F-1 overexpression on the proliferation of human gastric carcinoma cell line MKN-45. METHODS： The fragment of E2F-1 gene was amplified by polymerase chain reaction （PCR）. A eukaryotic vector expressing E2F-1 （pCMV-E2F1-HA） was constructed with pCMV-HA, and transfected into COS-7 cells to identify whether E2F-1 was successfully subcloned into the eukaryotic vector. pCMV-E2F1-HA was transfected into MKN-45 cells. The expression of E2F-1 in MKN-45 cells was detected by Western blot. The proliferation of MKN-45 cells was observed by colony formation assay and MTS method. Cell cycle was analyzed by flow cytometry （FCM）. RESULTS： Recombinant plasmid pCMV-E2F1-HA was successfully constructed and transfected into COS-7 and MKN-45 cells. MKN-45 cell clones were significantly fewer in pCMV-E2F1-HA group than in pCMV-HA group （4.7±1.8 vs. 30.2±6.7, P〈0.01）. The proliferation rate of MKN-45 cells was significantly lower in pCMV-E2F1-HA group （73.5% on Day 5, 63.5% on Day 6, 56.1% on Day 7） than in pCMV-HA group （100%, P〈0.01）. The G0/G1 phase proportion of MKN-45 cells was significantly higher in pCMV-E2F1-HA group than in pCMV-HA group （71.4% vs. 59.7%, P〈0.05）; the S phase proportion was significantly lower in pCMV-E2F1-HA group than in pCMV-HA group （18.7% vs. 30.7%, P〈0.05）. CONCLUSION： Overexpression of E2F-1 suppresses the proliferation of gastric cancer MKN-45 cells.

关 键 词：胃肿瘤 MKN-45细胞 E2F-1 转染 增殖 

分 类 号：R735.2[医药卫生—肿瘤]