中文摘要：

目的：构建腺病毒表达载体Ad-TCF4和Ad-ΔTCF4（缺失了β-catenin结合位点）,并初步鉴定其在间充质干细胞（mesenchymal stem cells,MSCs）中的表达,为进一步研究Wnt/β-catenin信号通路在MSCs中的功能以及应用MSCs进行干细胞移植和基因治疗奠定基础。方法：以MSCs的cDNA为模板,扩增TCF4和ΔTCF4基因,将基因序列定向克隆至穿梭质粒载体pShuttle-CMV后,转化含pAdEasy-1的BJ5183感受态细胞进行同源重组,获得重组腺病毒载体Ad-TCF4和Ad-ΔTCF4。PCR以及PacⅠ酶切鉴定后,Ad-TCF4和Ad-ΔTCF4转染QBI-293A进行包装和扩增。检测重组载体的病毒滴度以及感染MSCs的最适病毒复数（multiplicity of infection,MOI）,并探讨Ad-TCF4和Ad-ΔTCF4在MSCs中的表达以及对迁移的影响。结果：成功构建Ad-TCF4和Ad-ΔTCF4,病毒滴度分别为1.1×107pfu/mL和1.3×107pfu/mL,150 MOI病毒感染MSCs的效率达到90%以上;蛋白质印迹和TOPFlash/FOPFlash报告基因检测显示TCF4在MSCs中表达增加,并具有调节下游靶基因转录的活性;Boyden小室迁移实验发现感染Ad-ΔTCF4能显著抑制MSCs向LiCl的迁移能力,而感染Ad-TCF4并不能提高MSCs的迁移能力。结论：成功构建Ad-TCF4和Ad-ΔTCF4,能高效感染MSCs并正确表达。

英文摘要：

Objective： To construct recombinant adenovirus encoding full-length T cell factor 4（TCF4） and a mutant TCF4（ΔTCF4）,which delete the binding sites of β-catenin,and to investigate the expression and activity of these recombinant adenoviruses in MSCs,thereby providing the foundation for further study about the role of Wnt / β-catenin signaling in MSCs and sheding a light on the usefulness of MSCs in cell transplantation and gene therapies.Methods： The DNA sequences of TCF4 and ΔTCF4 were amplified from cDNA of MSCs by RT-PCR,and were subcloned into the shuttle plasmid pAdtrack-CMV,forming transfer vectors pAdtrack-CMV-TCF4 and pAdtrack-CMV-ΔTCF4.The plasmids were recombined in BJ5183 that has transformed by adenoviral backbone plasmid pAdEasy-1.Subsequently,both recombinant plasmids of Ad-TCF4 and Ad-ΔTCF4 were confirmed by RT-PCR,PacⅠ digesting analysis,and DNA sequencing.The identified Ad-TCF4 and Ad-ΔTCF4 were transfected to QBI-293A cells to produce the adenoviruses.To purify and titrate of recombinant adenoviral vectors,and to discuss Ad-TCF4 and Ad-ΔTCF4 vectors infected MSCs.Western blotting was applied to detect the expression of TCF4 in MSCs infected with Ad-TCF4.Luciferase activity assay was used to measure the activity of Ad-ΔTCF4 in MSCs.Results： The adenovirus vectors encoding target genes were proved to be recombined successfully in QBI 293A;and their titer virus were 1.1 × 107 pfu / mL and 1.3 × 107 pfu / mL respectively,The optimal virus of Ad-TCF4 and Ad-ΔTCF4 were 150 MOI,in which the efficiency of infection reached more than 90%.Western blot analysed the high expression of TCF4 in Ad-TCF4-infected MSCs,while luciferase activity was effectively suppressed in MSCs infected with Ad-ΔTCF4.MSCs infected with Ad-ΔTCF4 displayed decreased migration as compared with the control,however,infection with Ad-TCF4 showed no obvious differences.Conclusion： The recombinant adenoviruses encoding TCF4 and ΔTCF4 were successfully constructed and could infect MSCs efficiently.