显性负性突变型TGF-β Ⅰ型受体腺病毒的构建和功能验证

Construction and validation of adenovirus expressing dominant negative TGF-β type Ⅰ receptors

目的:构建制备显性负性突变型转化生长因子(TGF-β)Ⅰ型受体腺病毒,并验证其对于相应配体功能的抑制效应.方法:利用分子克隆技术构建和重组腺病毒技术,构建制备显性负性突变型TGF-βⅠ型受体腺病毒,并用其感染C3H10细胞,采用荧光显微镜观察和RT-PCR证明显性负性突变型TGF-βⅠ型受体腺病毒在C3H10细胞中的表达.采用碱性磷酸酶(ALP)定量和荧光素酶报告基因实验,分析显性负性突变型TGF-βⅠ型受体腺病毒对骨形成蛋白2(BMP2),TGF-β1功能的影响,证明其抑制相应配体功能的有效性.结果:构建和制备了显性负性突变型TGF-βⅠ型受体腺病毒,其可以感染C3H10细胞并在C3H10细胞中表达相应的显性负性TGF-βⅠ型受体;特定的显性负性突变型TGF-βⅠ型受体腺病毒可分别抑制由BMP2诱导的ALP活性及由TGF-β1诱导的荧光素酶活性.结论:成功构建制备的7种显性负性突变型TGF-βⅠ型受体腺病毒具有抑制相应配体功能的特性,为分析TGF-βⅠ型受体功能及靶向TGF-β信号通路的基因治疗提供了有用的工具.

AIM： To construct recombinant adenovirus containing dominant negative TGF-β type I receptors and to validate its inhibitory effect on the ligand＇s function. METHODS： The adenovirus containing dominant negative TGF-β type I receptors is constructed using molecular clone and recombinant adenovirus assay. C3H10 cells were then infected by the adenovirus and the expression of adenovirus in C3H10 cells was confirmed by fluorescence microscope and RT-PCR. Alkaline phosphatase quantitative assay and luciferase reporter assay were used to analyze the inhibitory effect of the adenovirus on BMP2-induced ALP activity and TGF-β-induced luciferase activity. RESULTS： The adenovirus containing dominant negative TGF-β type I receptors infected C3H10 cells and expressed dominant negative TGF-β type I receptors in C3H10 cells. Adenovirus expressing dominant negative TGF-β type I receptors inhibited the BMP2-induced ALP activity and TGF-β-indueed lueiferase activity. CONCLUSION： The adenovirus containing dominant negative TGF-β type I receptors is successfully constructed and its inhibitive effect on the ligand＇s function is confirmed, which provides a useful approach to further studying the functions of TGF-β type I receptors and to targeting TGF-β signaling for the disease therapy.