中文摘要：

目的：构建E2F-1真核表达载体，建立稳定过表达E2F-1的胃癌细胞株，探讨E2F-1基因过表达对胃癌MGC-803细胞化疗敏感性的影响。方法：应用Trizol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Ec,oRI和Hindm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—timequantitativePCR）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／E2F-1组（实验纽）、MGC-803／EV纽（阴性对照纽）和MGC-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EcoRⅠ和HindⅢ双酶切后得到预期片段，测序结果与报道的E2F-1基因之ORF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Westemblotting实验证实重组载体pCMV—E2F-1-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-FU）、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。

英文摘要：

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