转化生长因子β/骨形成蛋白应答元件表达质粒的构建及功能检测  

Construction and functional analysis of plasmids containing TGF-β/BMP responsive elements for regulating reporter gene expression

作　　者：胡青婷[1,2] 沈旭[1,2] 张凯[1,2,3] 贾岽 艾文兵[3] 张艳琼[1,2] 柳长柏[1,2] 吴江锋[1,2] 

机构地区：[1]三峡大学医学院,宜昌443002 [2]三峡大学分子生物学研究所,宜昌443002 [3]三峡大学第二临床医学院,宜昌443002

出　　处：《解剖学杂志》2014年第5期625-628,共4页Chinese Journal of Anatomy

基　　金：国家自然科学基金（81170412;81070348;81200307）;湖北省卫生厅科研指导性项目（JX6C-26）

摘　　要：目的：为了确认与转化生长因子β/骨形成蛋白（TGF-β/BMP）信号通路相关的转录因子,建立靶向治疗肝纤维化的筛选平台.方法：通过分子生物学技术设计、合成TGF-β/BMP应答元件（TRE/BRE）,构建真核表达质粒pGLuc-TRE-MiniTK、pGLuc（BRE）2-MiniTK和pGLuc（GCCG） 9-MiniTK.通过脂质体将pGLuc-TRE-MiniTK转染到小鼠成纤维细胞L929内,并采用TGF-β的持续活化型受体CA-ALK5激活TGF-β信号;将p-GLuc（BRE）2-MiniTK和pGLuc-（GCCG）9-MiniTK分别转染到大鼠肝星状细胞HSC-T6内,用BMP的持续活化型受体CA-ALK3激活BMP信号.然后,通过荧光检测培养细胞上清中的GLuc表达量,以此确认TGF-β/BMP应答元件的功能.结果：酶切鉴定和测序分析证实TRE、（BRE）2及（GCCG）9的碱基序列、插入方向和阅读框架正确.荧光素酶检测显示TRE和（BRE）2的荧光素酶GLuc大量表达,而（GCCG）9的荧光素酶GLuc表达不理想.结论：真核表达质粒pGLuc-TRE-MiniTK和pGLuc-（BRE） 2-MiniTK具备筛选与TGF-β/BMP信号通路相关转录因子的功能.Objective： To confirm transcription factors which are related to TGF-β/BMP signaling pathways, we established the therapy hepatic fibrosis screening platform with selecting intervention targeted therapy molecular. Methods： TGF-β/BMP responsive elements, eukaryotic expression plasmids pGLuc-TRE-MiniTK, pGLuc-（BRE）2-MiniTK and pGLuc-（GCCG）9- MiniTK, were designed and synthesized by molecular biology techniques. CA-ALK5 activated TGF-β signaling pathway in the mouse L929 when plasmids pGLuc-TRE-MiniTK were transfected into these cells with FuGENNE@ HD Transfection Regent; CA-ALK3 activated BMP signaling pathway in the rat HSC-T6 when plasmids pGLuc-（CK2CG）9-MiniTK and pGLuc-（BRE）2- MiniTK were transfected into these cells with FuGENNE@ HD Transfection Regent respectively. The function of TGF-β/ BMP responsive elements were analysed by Luciferase Reporter Gene Assay. Results： The eukaryotic expression plasmids containing TGF-β/BMP responsive elements have been constructed successfully after having been identified by enzyme digestion and DNA sequencing analysis; A lot of GLuc of pGLuc-TRE-MiniTK and pGLuc-（BRE）2-MiniTK were expressed by luciferase assay, but the expressions of GLuc of pGLuc-（GCCG）9-MiniTK were not too much. Conclusion： The eukaryotic expression plasmids pGLuc-TRE-MiniTK and pGLuc-（BRE）2-MiniTK have the functions as selecting transcription factors in related with TGF-β/BMP signaling pathway successfully.

关 键 词：转化生长因子β应答元件 骨形成蛋白应答元件 肝纤维化 

分 类 号：R619.6[医药卫生—外科学]