构建人三叶因子3腺病毒载体对胆管上皮细胞迁移的调控作用

Recombinant adenovirus containing human TFF3 gene regulates migration in human biliary epithelial cells

目的构建含有人三叶因子3(trefoil factor family 3,TFF3)基因的腺病毒载体,并观察其对胆管上皮细胞迁移的调控作用。方法利用PCR技术对目的基因TFF3全长进行扩增,测序后亚克隆至腺病毒穿梭质粒构建载体pAdTrack-CMV-TFF3,转化至含腺病毒骨架质粒pAd-Easy的大肠杆菌株BJ5183感受态细胞中进行同源重组,筛选重组子并经内切酶PacⅠ线性化,转染293细胞进行病毒包装及扩增,并进行滴度测定。构建的TFF3腺病毒载体感染人胆管上皮细胞后用RT-PCR及Western blot检测目的基因表达水平,并进行划痕实验观察细胞迁移能力的变化。结果成功扩增了大小约为458 bp的人TFF3基因,测序报告显示目的基因序列与NCBI报道一致;成功构建了TFF3的同源重组腺病毒穿梭载体,经293细胞包装及扩增的病毒滴度为1.3×109CPU/mL。RT-PCR及Western blot结果显示转染人胆管上皮细胞后其TFF3表达明显增强,细胞迁移能力也明显高于对照组。结论成功构建含TFF3基因的腺病毒载体,为进一步研究TFF3在胆管上皮损伤修复中的作用奠定了基础。

Objective To construct a recombinant adenovirus vector of human trefoil factor family 3 （TFF3） and to determine its effect on the migration in human biliary epithelial cells. Methods The genomie fragment of target gene TFF3 was amplified with PCR with genomic DNA of liver cancer tissue as template. The obtained product was subcloned into adenovirus shuttle plasmid to construct vector pAdTrack-CMV-TFF3 after sequencing. Homologous recombination was performed by transfering the vector to E. coli BJ5183 containing the backbone pAd-Easy. The correct recombinant pAdEasy-TFF3 was selected and linearized with Pac I , then transfected into 293 cells for packaging and amplification. The titration of the recombinant adenovirus was deter- mined. RT-PCR and Western blotting were used to determine the expression of TFF3 at mRNA and protein lev- els in human biliary epithelial cells after transfection. Finally, cell scratch test was performed to observe the effect on cell migration before and after transfection. Results The target fragment of TFF3 with 458 bp was successfully amplified, which was completely the same as the sequence published GenBank by DNA sequen- cing. The TFF3 homologous recombination adenovirus vector was successfully constructed with a titer of 1.3 × 109CPU/mL. RT-PCR and Western blotting indicated significant increased expression of TFF3 at mRNA and protein levels in human biliary epithelial cells after transfection. Cell scratch test showed that cell migration was obviously activated and enhanced after transfeetion. Conclusion Recombinant adenovirus vector containing TFF3 gene is successfully constructed, which provides a satisfactory transduction vector to research the role of TFF3 in human biliarv enithelium wound h^linc~