婴儿双歧杆菌介导的sKDR原核克隆表达及其对血管内皮细胞增殖的影响  

Cloning and Prokaryotic Expression of Bifidobacterium Infantis-mediated sKDR and Its Effect on Proliferation of Vascular Endothelial Cells

作　　者：毛淑华[1] 姬玲玲[1] 刘洪[1] 徐一洲[1] 黄煌[1] 黄英[1] 易成[2] 

机构地区：[1]四川大学华西基础医学与法医学院病理生理学教研室,成都610041 [2]四川大学华西医院腹部肿瘤科

出　　处：《四川大学学报（医学版）》2009年第5期784-786,802,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30570693);四川省科技厅攻关计划科研基金(批准号2006Z09-030)资助

摘　　要：目的构建婴儿双歧杆菌介导的sKDR基因肿瘤靶向性转运系统并观察其对血管内皮细胞增殖的影响。方法PCR扩增sKDR基因,提取与纯化pET32a质粒。EcoRⅠ和XhoⅠ对sKDR基因和pET32a质粒分别进行双酶切,用T4DNA连接酶将目的基因片段和载体连接。电穿孔法将连接反应物转染婴儿双歧杆菌。厌氧培养重组质粒转化菌,并将其处理液加入由VEGF诱导的人脐静脉内皮细胞(HUVECs)培养基中培养24h,MTT比色法测定细胞存活率。结果PCR、酶切鉴定及核酸测序结果表明,获得了含pET32a-sKDR重组质粒的婴儿双歧杆菌;RT-PCR及SDS-PAGE电泳结果证实sKDR在基因及蛋白水平成功表达。与空质粒对照组相比,重组质粒转化菌处理液可明显抑制HUVECs增殖(P<0.01)。结论成功构建了婴儿双歧杆菌介导的sKDR基因转运系统,体外实验证实其可明显抑制血管内皮细胞的增殖。Objective To construct Bifidobacterium Infantis-mediated sKDR gene transferring system and to investigate its effect on the proliferation of vascular endothelial cells. Methods sKDR gene amplified through PCR, and pET32a plasmid extracted from E. coli JM109 were digested respectively by two kinds of restriction enzyme （EcoR Ⅰ and Xho Ⅰ ） and then were connected by T4 DNA Ligase. Finally, the recombinant plasmid was transformed into Bifidobacterium Infantis by electroporation. Human umbilicus vein endothelial cells （HUVECs） were cultivated in the nutritive media containing the extract of positive transformed bacteria for 24 h. The cell viability was analyzed with MTT assay. Results The positive transformed Bifidobacteriurn Infantis with recombinant pET32a-sKDR plasmid was established and could express sKDR at the levels of gene and protein. Compared with the untreated group, the proliferation of HUVECs cultivated with the extract of positive transformed bacteria was inhibited significantly （P〈0.01）. Conclusion The Bifidobacterium Infantis-mediated sKDR gene transferring system was constructed successfully and it could remarkably inhibit the proliferation of vascular endothelial cells.

关 键 词：婴儿双歧杆菌 抗血管生成 血管内皮生长因子(VEGF) 可溶性血管内皮生长因子受体 (sKDR) 血管内皮细胞 

分 类 号：R73-3[医药卫生—肿瘤]