重组腺病毒介导的人内皮抑素基因在SW1990细胞中的表达  

Expression of human endostatin gene in SW1990 cell line mediated by recombinant adenovirus

作　　者：吕纯业[1] 胡先贵[1] 邵成浩[1] 刘瑞[1] 张怡杰[1] 金钢[1] 

机构地区：[1]第二军医大学附属长海医院普三科,上海200433

出　　处：《中华实验外科杂志》2007年第10期1220-1222,共3页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金(30200275);上海市青年科技启明星计划资助项目(05QMX1472)

摘　　要：目的应用AdEasy载体系统构建含人内皮抑素(hEndostatin)的重组腺病毒载体,观察感染后hEndostatin在SW1990中的表达和生物学活性。方法将hEndostatin克隆至穿梭质粒pShuttle-CMV,与腺病毒骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,获得腺病毒重组质粒,腺病毒重组质粒转染293细胞进行包装,获取hEndostatin重组腺病毒。同法构建重组腺病毒Ad-LacZ,转染SW1990细胞,确定重组腺病毒的最适感染复数(MOI),hEndostatin重组腺病毒以最适MOI转染SW1990细胞,观察转染后1-7 d hEndostatin的表达及生物学活性。结果成功构建了hEndostatin重组腺病毒,该腺病毒可高效介导hEndostatin在SW1990细胞中的表达,MOI=100为最适感染复数;转染后1～7 d,上清中hEndostatin蛋白的浓度分别为(1.6±0.3)、(8.5±0.6)、(54.3±4.4)、(256.9±25.8)、(596.6±38.7)、(321.7±21.2)、(132.6±7.6)μg/L;表达产物具有生物学活性,显著抑制血管内皮细胞的增殖、迁移(P<0.01)。结论重组腺病毒可介导hEndostatin基因在SW1990细胞中的有效表达。To construct a human endostatin adenovirus vector and observe the expression and bioacfivity of endostatin in SW1990 cell line after recombinant adenovirus infection. Methods Human endostain cDNA was cloned into shutde vector pShutde-CMV, and the resultant plasmid was linearized and subsequently cotransformed into E. coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1. Recombinants were then selected and the linearized recombinant plasmid was transfected into 293 cell line to produce recombinant adenovirus. Endostatin recombinant adenovirus was confirmed by PCR and Western blot analysis. Recombinant adenovirus Ad-Lac Z constructed as the same steps was used to infect SW1990 cells at different MOI to find its optimal MOI. SW1990 cells were infected by endostatin adenovirus at the optimal MOI, and the endostatin protein concentration and bioactivity in supernatant were detected every day within a week. Results A human endostatin recombinant adenovirus vector was constructed successfully and could infect SW1990 cell hne with high efficiency. MOI = 100 was the optimal multiplicity of infection. During 1-7 days after transfection, endostatin protein concentration in the supematantofSW1990was （1.6±0.3）, （8.5±0.6）, （54.3±4.4）, （256.9±25.8）, （596.6± 38.7）, （321.7 ±21.2） and （ 132.6±7.6） μg/L respectively. This endostatin protein significantly inhibited the proliferation and migration of human umbihcal vein endothelial cells （ P 〈 0.01 ）. Conclusion Recombinant adenovirus can mediate efficient and effective expression of human endostatin in SW1990 cells.

关 键 词：胰腺肿瘤 腺病毒 内皮抑素 基因表达 

分 类 号：R735.9[医药卫生—肿瘤]