中文摘要：

目的：构建沉默结缔组织生长因子（connective tissue growth factor,CTGF）基因的复制缺陷型重组腺病毒Ad-siCTGF,并进行功能验证。方法：选取已验证的能高效沉默CTGF基因的靶序列,克隆入载体pSES-HUS中;将质粒线性化处理后与腺病毒骨架质粒pAdEasy共同转染大肠埃希菌BJ5183,构建重组腺病毒载体Ad-siCTGF;腺病毒载体用PacI酶切线性化处理后转染HEK293细胞,包装重组腺病毒;采用＂乒乓交互感染法＂提高病毒滴度。用此病毒感染4T1细胞,行实时荧光定量PCR（real-time fluorescence quantitative-PCR,RFQ-PCR）及Western印迹法验证其沉默效果。结果：PacI酶切电泳证实重组腺病毒载体Ad-siCTGF构建成功,扩增纯化后测定重组病毒Ad-siCTGF的滴度为2.6×10^10pfu/mL。感染此病毒后,4T1细胞中CTGF mRNA表达水平下调至36.27%,蛋白表达水平下调至31.56%。结论：成功构建能沉默CTGF基因的重组腺病毒,为进一步研究CTGF在肿瘤中的作用机制奠定了基础。

英文摘要：

Objective：To design and construct the replication-deficient recombinant adenovirus Ad-siCTGF which can silence the expression of connective tissue growth factor（CTGF） by RNA interference and verified its function.Methods：A specific sequence,which was verified to be able to silence CTGF gene with high efficiency,was cloned into pSES-HUS vector to produce the shuttle plasmid pSES-siCTGF.The plasmid after Pme Ⅰ linearization was cotransduced with pAdEasy into BJ5183 E.coli strains to construct recombinant vector Ad-siCTGF.After linearization treatment with Pac Ⅰ enzyme digestion Ad-siCTGF was transfected into HEK293 cells via liposome mediation.The recombinant adenovirus was packaged.The titer of the Ad-siCTGF was increased after three times of cross-infection.4T1 cells were infected with the adenovirus.The silencing efficiency was tested by real-time fluorescence quantitative（RFQ）-PCR and Western blotting.Results：Pac Ⅰ enzyme digestion electrophoresis indentified that recombinant adenovirus was successfully constructed.The titer of the recombinant adenovirus Ad-siCTGF was 2.6×10^10 pfu/mL after amplification and purification.The CTGF mRNA and protein expression in 4T1 cells were decreased by 36.27% and 31.56%,respectively,compared with the control groups.Conclusion：The recombinant adenovirus which can silence the expression of CTGF was successfully constructed.It laid a good foundation for further investigation of the action mechanism of CTGF in tumor cells.