腺病毒介导人肠三叶因子基因对肠上皮细胞的影响  

Effect of adenovirus mediated human intestinal trefoil factor gene on intestinal epithelial cells

作　　者：孙勇[1] 勒娟[1] 陈宝君[1] 潘晓峰[1] 张方[1] 

机构地区：[1]徐州医学院附属淮海医院烧伤科 中国人民解放军第九七医院烧伤科,江苏221004

出　　处：《中华实验外科杂志》2015年第4期738-742,共5页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81100252）;南京军区医学科研课题重点项目（12Z10）

摘　　要：目的 构建含有人肠三叶因子（hITF）基因的腺病毒载体,并观察其对肠上皮细胞的影响.方法 首先构建重组腺病毒载体,并感染人肠上皮细胞株Caco-2;然后行反转录-聚合酶链反应（RT-PCR）及Western blot检测,从mNRA及蛋白水平检测hITF表达;感染病毒细胞做爬片后行细胞免疫荧光检查;最后通过划痕实验,检测重组腺病毒对肠上皮细胞迁移功能的影响.结果 成功构建出重组腺病毒,可以感染人肠上皮细胞株Caco-2,感染效率接近100％;RT-PCR和Western blot检测证实,hITF被成功转录并分泌表达;细胞免疫荧光显示,hITF可表达于细胞核和细胞质,但以细胞质为主;划痕实验证明感染重组腺病毒对肠上皮细胞具有很强的促迁移能力,重组腺病毒组迁移距离为（216.67±16.43） μm,显著高于空载病毒组的（68.00±8.37） μm和对照组的（68.00±8.37）μm.结论 成功构建了含hITF基因的腺病毒载体,该载体对肠上皮细胞具有促迁移作用,为hITF基因治疗的应用打下基础.Objective To construct a recombinant adenovirus vector of human intestinal trefoil factor （hITF） and observe its effect on human intestinal epithelial cells （Caco-2）.Methods A recombinant adenovirus vector containing hITF gene was constructed and infected Caco-2 cells.The expression of hITF mRNA and protein were detected by reverse transcriotion-polymerase chain reaction （RT-PCR）,Western blotting and immunofluorescence.Then,wounds were established in confluent monolayers of Caco-2 ceils infected with recombinant adenovirus vector,and the width of the cells migration was measured in a blind manner.Results The recombinant adenovirus vector Ad-hITF was constructed and the infection efficiency was about 100%.RT-PCR and Western blotting assay demonstrated that hITF was transcribed and expressed successfully.hITF was proved to distribute in cytoplasm by immunofluorescence assay.In addition,Ad-hITF enhanced migration activity of HT-29 cells obviously.Conclusion Recombinant adenovirus vector containing hITF gene was successfully constructed and its effect was confirmed in promoting cells migration.This research lay foundations for the further application of hITF gene therapy.

关 键 词：肠三叶因子 腺病毒 肠上皮细胞 迁移 

分 类 号：R644[医药卫生—外科学]