Generation of an anti-fibrotic therapeutic gene using a tubular epithelium-specific expression gene casette and iKB cDNA

为了确定骨桥蛋白(OPN)基因启动子中的肾小管上皮细胞特异的正调控元件，我们构建了含有OPN启动子不同片段的荧光素酶报告基因，并利用小鼠肾小管上皮细胞(MPTEC)进行了瞬时转染实验。除mPTEC外，还利用成纤维细胞(3T3)和单核细胞(RAW264)进行了转染实验。OPN启动子的-600~-825bp片段在mPTEC中呈正转录活性，而在3T3和RAW264中不呈正转录活性。反之，在重组转化生长因子-b1处理后，mPTEC、3T3和RAW264中的-115~-189bp片段显示出正转录活性。因此，-600到-825bp的片段被认为含有肾小管上皮细胞特异的、阳性的调节元件。我们现在正在利用该片段构建肾小管上皮细胞特异性表达基因盒，并在体外和体内测试IKB在肾小管上皮细胞特异性表达是否可以通过下调核因子-kB的活性来减轻纤维化。

To identify tubular epithelium-specific, positive regulatory elements in the promoter of osteopontin (OPN) gene, we generated luciferase reporter gene constructs bearing various fragments of the OPN promoter and performed transient transfection experiments using murine tubular epithelial cells (mPTEC). In addition to mPTEC, fibroblasts (3T3) and monocytes (RAW264) were also employed for the transfection experiments. The -600 to -825 bp fragment of the OPN promoter constitutively showed positive transcription activity in mPTEC, but not in 3T3 and RAW264. In contrast, the -115 to -189 bp fragment showed positive transcription activity in mPTEC as well as 3T3 and RAW264 in response to recombinant transforming growth factor-b1 treatment. Thus, the -600 to -825 bp fragment is suggested to contain tubular epithelium-specific, positive regulatory elements. We are now generating tubular epithelium-specific expression gene casette using this fragment, and testing whether tubular epithelium-specific expression of iKB can attenuate fibrosis through down-regulation of NF-kB activity in vitro and in vivo.