【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-TREMiniTK、p-GLuc（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。更多还原

【Abstract】 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-（GCCG）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.更多还原