E2F-1基因过表达影响胃癌MGC-803细胞对化疗药物敏感性的研究  

The Effects of E2F-10verexpression on Chemosensitivity of Gastric Cancer Cell Line MGC-803

作　　者：钱倩[1] 王长青[1] 李雷[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,南宁市530021 [2]广西医科大学第一附属医院麻醉科,南宁市530021

出　　处：《中国肿瘤临床》2009年第16期945-947,956,共4页Chinese Journal of Clinical Oncology

基　　金：国家自然科学基金(编号:30860273);广西医疗卫生重点科研项目基金资助(编号:桂卫重200844 )~~

摘　　要：目的:构建E2F-1真核表达载体,建立稳定过表达E2F-l的胃癌细胞株,探讨E2F-l基因过表达对胃癌MCC-803细胞化疗敏感性的影响方法:应用Tri zol法从胃癌的癌组织中提取mRNA逆转录合成cDNA,按Gene-Bank公布的E2F-1基因序列设计带有酶切位点的引物并行扩增获得E2F-1的ORF片段;然后经限制性内切酶EcoR l和Hind Ⅲ双酶切后将E2F-1定向克隆到真核表达载体pCMV-HA2中转化筛选用脂质体Lipofectamine2000将该载体转染MGC-803胃癌细胞,然后用含G418的培养基筛选获得具Genectin抗性的过表达E2F-1的胃癌细胞株实时定量PCR( real-time quantitative PCR)和蛋白印迹(Western blotting)技术检测MGC-803/E2F-1细胞内E2F-1表达情况四唑蓝(MTT)法检测MGC-803/E2 F-1组(实验组)、MGC-803/EV组(阴性对照组)和MCC-803组(未转染组)细胞对化疗药物的敏感性结果:重组载体pCMV-E2F-1-HA2经EcoR I和Hind Ⅲ双酶切后得到预期片段,测序结果与报道的E2F-1基因之0RF完全一致,证明目的基因已成功克隆入真核表达载体,获取具Genectin抗性的细胞克隆、并进行了扩增。RT-PCR和Western blotting实验证实重组载体pCMV-E2F-l-HA2成功转入胃癌细胞内,并稳定过表达E2F-1 MTT法检测发现,5-氟尿嘧啶(5-FL)、阿霉素(ADM)、丝裂霉素(MMC)对MGC-803/E2F、-1组细胞的抑制率分别为71.45%、74.03%、76.72%,均明显高于MGC-803/EV组(45.58%、53.69%、54.58%)和MGC-803组(53.70%、54.67%、55.05%) (P<0.05)结论:成功构建了真核表达载体pCMV-E2F-1-HA2,并建立了稳定过表达E2F-1的胃癌细胞株MGC-803/E2F-1。E2F-1过表达增强了胃癌细胞对化疗药物的敏感性。Objective： To investigate the effects of E2F-1 overexpression on chemosensitivity of gastric cancer cell line MGC-803, we constructed a eukaryotic expression vector containing the E2F-1 gene and then transfected it into gastric cancer cell line MGC-803. Methods： We extracted mRNA from human gastric cancer tissue and synthesized cDNA by reverse transcription. Using the E2F-1 gene sequence in GeneBank, we designed a primer that contained restriction enzyme sites and amplified the ORF of E2F-1. After double digestion with restriction enzymes EcoRI and Hind ]I[, the products were cloned into the eukaryotic expression vec- tor pCMV-HA2 by directional cloning, transformation and screening. The recombinant vector was transfected into gastric cancer cell line MGC-803 with Lipofectamine 2000 and selected for in medium containing G418 to obtain clones that overexpressed E2F-1 and had resistance to Genetecin. The expression of E2F-Ⅲ mRNA and protein was detected by real-time quantitative polymerase chain reaction （RT-PCR） and Western blotting methods, respectively. We evaluated chemotherapeutic drug sensitivity of MGC-803/E2F-1 cells （experimental group）, MGC-803/EV cells （control group） and MGC-803 cells （untransfected group） by methyl thiazolyl tetrazolium （MTT） assay. Results： After the recombinant vector pCMV-E2F-1-HA2 was double digested with EcolR Ⅰ and Hind Ⅲ, the individual fragments were obtained. The sequencing results were consistent with the reported E2F-1 gene sequence, indicating that the E2F-1 gene fragment had been successfully cloned in- to the eukaryotic expression vector. Cell clones with resistance to Genetecin were obtained and amplified. Experimental results of RT-PCR and Western blotting confirmed that the recombinant vector pCMV-E2F-1-HA2 was successfully transfected into gastric cancer cell line MGC-803 and stably overexpressed the E2F-1 gene. In the MTT assay, the inhibition rates Of 5-fluorouracil （5-Fu）, adriamycin （AMC） and mitomycin （MMC） in MGC-803/E

关 键 词：E2F-1 转染 MGC-803胃癌细胞 药敏试验 

分 类 号：R735.2[医药卫生—肿瘤] R737.9[医药卫生—临床医学]