CAJ | 학술논문

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.
목적 구건함유인장삼협인자(hITF)기인적선병독재체,병관찰기대장상피세포적영향.방법 수선구건중조선병독재체,병감염인장상피세포주Caco-2;연후행반전록-취합매련반응(RT-PCR)급Western blot검측,종mNRA급단백수평검측hITF표체;감염병독세포주파편후행세포면역형광검사;최후통과화흔실험,검측중조선병독대장상피세포천이공능적영향.결과 성공구건출중조선병독,가이감염인장상피세포주Caco-2,감염효솔접근100％;RT-PCR화Western blot검측증실,hITF피성공전록병분비표체;세포면역형광현시,hITF가표체우세포핵화세포질,단이세포질위주;화흔실험증명감염중조선병독대장상피세포구유흔강적촉천이능력,중조선병독조천이거리위(216.67±16.43) μm,현저고우공재병독조적(68.00±8.37) μm화대조조적(68.00±8.37)μm.결론 성공구건료함hITF기인적선병독재체,해재체대장상피세포구유촉천이작용,위hITF기인치료적응용타하기출.
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.