非肌肉肌球蛋白II通过mTOR通路调节肝星状细胞迁移的机制研究

The targeted migration of hepatic stellate cells(HSCs) is one of the most important mechanisms in the development of liver fibrosis. Nonmuscle myosin II involves in the process of cell contratction and migration,and it can also modulates the EGFR-dependent activation of AKT,which is the upstream signaling molecular of mTOR. mTOR pathway takes part in the regulation of many types of cells' migration, including HSCs. Our preliminary GeneChip Analysis on human liver tissue showed that mTOR signaling pathway lies in the networks of liver fibrosis differentiated genes.And immunohistochemistry staining demonstrates that the expression of mTOR protein is up-regulated in the liver of CCl4-treated mouse.In order to explore the mechanism of NMM II and mTOR in the hepatic fibrogenesis in chronic liver injury，this project intends to research the impact of down-regulation mTOR signaling and NMM II by siRNA gene silencing technologies and by administation of NMM II inhibitor blebbistatin and mTOR inhibitor rapamysin on the activation, proliferation, migration, contraction of HSCs from cell levels. The interventiaon of mTOR inhibitor rapamysin and Ad-siRNA-NMM II on rat HSCs and the extent of liver fibrosis will be explored from animal level. From different levels，we will clarify the roles and mechanisms of mTOR in the migration of hepatic stellate cells under the regulation of nonmuscle myosin II, and provide novel therapeutic targets for the prevention and curation of liver fibrosis.

肝星状细胞（HSCs）的靶向迁移是肝纤维化发生及发展的重要机制。非肌肉肌球蛋白II（NMM II）参与细胞收缩和迁移，并可调节表皮生长因子受体依赖的mTOR上游分子AKT等信号分子活化。mTOR通路参与调控包括HSCs在内的多种细胞迁移。我们前期肝组织基因芯片分析显示mTOR通路处于肝纤维化差异基因网络中。免疫组化染色证实大鼠肝纤维化模型中存在mTOR蛋白表达增加。本课题拟从细胞水平利用siRNA基因静默、化学干预等方法研究下调NMM II表达对HSCs中mTOR通路及对HSCs迁移、收缩力的影响；并从动物水平探究NMM II腺病毒载体基因静默及mTOR抑制剂雷帕霉素对大鼠HSCs功能及肝纤维化程度的变化。从不同水平研究肝星状细胞迁移对肝纤维化进程的影响，阐明mTOR在NMM II调节HSCs迁移中的作用及机制，为今后肝纤维化的防治提供新的理论依据。

肝星状细胞（HSCs）的靶向迁移是肝纤维化发生及发展的重要机制。非肌肉肌球蛋白II（NMM II）参与细胞收缩和迁移，并可调节表皮生长因子受体依赖的mTOR上游分子AKT等信号分子活化。mTOR通路参与调控包括HSCs在内的多种细胞迁移。研究显示大鼠肝纤维化模型中存在mTOR蛋白表达增加，给于雷帕霉素干预后可下调mTOR表达，减轻肝纤维化程度。研究显示，大鼠肝星状细胞活化后，NMMII、S6K1、4EBP1基因表达量上升，NMMII和MTOR蛋白表达量上调；使用blebbistatin 、rapamycin及下调NMMII、mTOR基因表达均可抑制HSC细胞的迁移及收缩性；blebbistatin 、rapamycin、下调MTOR基因表达，可抑制HSC细胞增殖，NMMII基因下调对HSC增殖影响较弱。blebbistatin 、rapamycin干预及MTOR基因下调，可使细胞进入G2期速度减慢；NMMII基因下调对细胞周期无明显影响。blebbistatin 、rapamycin、下调MTOR、NMMII基因表达均对HSC细胞凋亡无影响。blebbistatin 、rapamycin可下调HSC细胞4EBP1、col1a1、col3a1基因影响较大；NMMII、MTOR基因下调可抑制S6K1表达。通过动物和细胞水平研究肝星状细胞迁移对肝纤维化进程的影响，阐明非肌肉肌球蛋白II对肝星状细胞迁移的调节作用是通过mTOR通路介导的，阻断mTOR通路、下调NMM II表达可减轻肝纤维化程度。为今后肝纤维化的防治提供新的理论依据。