人STAT3重组腺病毒的制备及其对胃癌细胞株SGC-7901增殖影响.docVIP

PAGE PAGE 10 人STAT3重组腺病毒的制备及其对胃癌细胞株SGC-7901增殖影响 董宗明1 赵永亮1 余佩武1Supported by the Natural Science Foundation of China ,Corresponding auther: Yu Peiwu,Tel:86-023 E-mail：yupeiwu01@[基金项目] 国家自然科学基金资助项目[通讯作者] Supported by the Natural Science Foundation of China ,Corresponding auther: Yu Peiwu,Tel:86-023 E-mail：yupeiwu01@ [基金项目] 国家自然科学基金资助项目 [通讯作者]余佩武，电话：023E-mail：yupeiwu01@ [摘要]：目的：制备可表达人转录因子STAT3的重组腺病毒，检测该基因在胃癌细胞株SGC-7901表达活性及对其增殖影响，为探讨STAT3信号通路在胃癌免疫抑制发生中的作用机制打下实验基础。方法：以人胎盘组织cDNAs为模板通过PCR获得hSTAT3完整阅读框，T/A克隆测序证实后，亚克隆至穿梭载体pAdTrack-CMV中，PmeI线性化与腺病毒骨架载体pAdEasy-1在BJ5183细菌同源重组，经Pac I线性化后转染293FT细胞包装，同时包装空载体腺病毒，进行病毒滴度测定。取AdV-STAT3、AdV-GFP分别感染SGC-7901细胞，设空白对照， Western-blot检测三组细胞STAT3蛋白表达，MTT法检测对细胞增殖的影响。结果：获得hSTAT3完整阅读框，T载体中测序无突变，亚克隆至pAdTrack-CMV，并与pAdEasy-1成功重组后包装AdV-STAT3，测定滴度为2.8×1010 pfu/ml，AdV-GFP滴度为3.2×1010 pfu/ml。与AdV-GFP组及空白对照组比，Western-blot示AdV-STAT3组蛋白明显表达，差异有统计学意义（P＜0.01），MTT示， AdV-1-STAT3组明显促进细胞增殖，差异有统计学意义（P＜0.01）。结论：首次成功制备hSTAT3的重组腺病毒，感染胃癌细胞株SGC-7901后高表达并促进其增殖，为进一步研究Jak2-STAT3信号通路与胃癌之间的关系奠定基础。 [关键词]：STAT3；重组腺病毒，胃癌，增殖 Construction of human STAT3 recombinant adenovirus vector and its effection on gastric cancer cell line SGC-7901 DONG Zongming 1ZHAO Yongliang1 Yu Peiwu1* HUAN Gang2 (1Department of General Surgery,Southwest Hospital,2Department of Medical Genetics, Third Military Medical University,Chongqing 40038,China) Abstract: The aim of this study is to prepare recombinant adenovirus vector containing human STAT3(signal transducer and activator of the transc ription 3) ,then examining its express in Gastric cell line SGC-7901 and its effect on proliferation of SGC-7901 , to lay the foundation for the further research of the relation between STAT3’s signal and the mechanism of the immunosuppression in gastric cancer. Methods: First the completely hSTAT3 code sequence was abtained by PCR using the cDNAs of placenta from healthy volunteer’s as the template ,next clone into pTA2 T vector and se