作用于SMAD3的RNA干扰的靶位点筛选、构建及其对肝纤维化作用研究

项目名称： 作用于SMAD3的RNA干扰的靶位点筛选、构建及其对肝纤维化作用研究

项目编号： No.30800979

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 郑素军

作者单位： 首都医科大学

项目金额： 20万元

中文摘要： 转化生长因子β65288;TGF-β65289;及激活素对肝星状细胞激活有重要作用，SMAD3是两者信号通路中共同关键蛋白。阻断SMAD3 蛋白表达可能起到高效抗肝纤维化。本研究项目做了以下工作：①#22312;体外，显示TGF-β#23545;大鼠肝星状细胞HSC-T6有促增殖、促肝纤维化相关胶原分泌等作用；HSC-T6保留了原代培养肝星状细胞部分特点，但并不完全相同。另一重要发现是大鼠肝细胞BRL-3A 对TGF-β#20419;凋亡和细胞周期阻滞作用并不敏感。②#35745;算机软件设计了SMAD3多靶位siRNA,于BRL-3A细胞进行了有效靶位点筛选，构建了3条以慢病毒载体为表达系统，可内源性高效表达的短发卡状SMAD3 shRNA；③#22312;体外，利用HSC-T6,发现shRNA慢病毒可有效阻断SMAD3 表达，有明显抗肝纤维化的作用。④MAD3shRNA在四氯化碳大鼠肝纤维化模型体内有明显抗肝纤维化作用。⑤#30740;究乙肝肝纤维化相关机制，发现磷酸鞘胺醇（S1P）及其受体（S1P receptor）促进肝星状细胞迁移，在人肝纤维化过程中有重要作用；MicroRNA-122 作用于HBx影响HBV复制

中文关键词： 肝纤维化；转化生长因子β65288;TGF-β65289;；SMAD3；短发卡状RNA（small hairpin RNA;shRNA）；慢病毒

英文摘要： Transforming growth factor-β65288;TGF-β65289;and Activin play the key role in activating hepatic stellate cells.SMAD3 is the common signal protein and the therapy of silencing the the expression of SMAD3 possiblely have the strong effect of anti-fibrosis .Our study have the following results①GF-βcan promote cell proliferation and the secretion of collagen by the rat HSC-T6 cell line. The results imply that the immortalized hepatic stellate cells recapture some features of primary HSCs, but differ significantly to the primary cells. Sensitivity of BRL-3A to TGF-βis low at the aspects of inducing apoptosis and cell cycle arrest.②ix pairs of siRNAs of rat smad3 were designed and synthesized and selected for their inhibition effect.Three recombinant lentivirus which express corresponding shRNA have been constructed successfully.③n vitro,shRNA can inhibit the expression of SMAD3 and showed the anti-fibrosis role. ④hRNA showed the obvious anti-fibrosis role in the rat fibrosis model induced by CCL4 injection. ⑤he exploration of the mechanism of HBV-related liver firosis in human showed that Sphingosine 1-phosphate (S1P)/S1P receptors plays an important role in human liver ?brosis and is involved in the directed migration of human hMFs into the damaged areas.miR - 122 may affected HBV replication by regulating the expression of HBx.

英文关键词： liver fibrosis；TGF-β65307;SMAD3；Small hairpin RNA;shRNA；Lentivirus