基于肝星状细胞激活和增殖调控的羊膜干细胞抗纤维化作用机制研究

Liver fibrosis are the most common liver disease in humans. In recent years, the incidence of liver fibrosis are increasing in China. Until now, there is still no effective treatments for liver fibrosis. Marrow stromal cells provide a promising strategy for the treatment of damaged tissues because of their selective migratory ability to injured cells. The human amniotic membrane stem cells (HAMSCs), with their attributes of high expansion potential, low immune response and no cytotoxicity or oncogenicity, showed the similar characteris of marrow stromal cells. In preliminary studies, we found that the HAMSCs can ameliorate liver fibrosis and supress the activation and proliferation of hepatic stellate cells (HSCs) through Notch signaling pathway. In this study, we will firstly determine the liver fibrosis tropic ability of HAMSCs by in vitro and in vivo migration assays and research the function of cytokine; Then, the HAMSCs and HSCs will indirect co-culture by transwell . The key cytokine which supress the activation and proliferation of HSCs cells will be analyzed by antibody microarry. Finally, the target gene of Notch signaling pathway and the mechanism of HAMSCs on supression the activation and proliferation of HSCs will be explored by modulating Notch signaling pathway. In general, HAMSCs may be a potential therapeutic means for treating liver fibrosis and clinical application. Results will contribute to understanding the molecular mechanisms of live fibrosis disease, and are helpful for the development of small molecule drugs for treatment of live fibrosis.

肝纤维化是最常见的肝脏疾病，其发病率在我国呈上升趋势，目前仍缺乏有效治疗手段。近年来间充质干细胞由于具有向损伤部位迁移并参与组织损伤修复的特性而引起了学术界的浓厚兴趣。羊膜干细胞具有间充质干细胞特性，且来源丰富、获取简便、无伦理道德争议、无致瘤性、免疫源性低。我们前期研究发现羊膜干细胞移植可通过Notch通路抑制肝星状细胞激活和增殖，发挥抗纤维化作用。本课题拟通过体外和体内研究明确羊膜干细胞对肝纤维化的趋向性，阐释各细胞因子在此过程中的作用；利用Transwell将羊膜干细胞与肝星状细胞共培养，抗体芯片技术鉴定羊膜干细胞抑制肝星状细胞激活和增殖的关键因子；利用过表达和SiRNA技术调控Notch相关信号通路，确定其靶基因，阐释羊膜干细胞抑制肝星状细胞激活和增殖的分子机制。本项目的实施可为肝纤维化的治疗提供一种可选方案，对进一步研究肝纤维化发病的分子机制，设计合理的治疗药物具有重要意义。

肝纤维化是最常见的肝脏疾病，持续的肝纤维化将破坏正常的肝脏结构，形成肝小结，改变血液循环，最终引起肝硬化，甚至演变成肝细胞癌。目前肝纤维化仍缺乏有效治疗手段。人羊膜间充质干细胞（hAMSCs）是近年来发现的一种新型干细胞，具有组织来源丰富、增殖能力强、无致瘤性、免疫源性低等优点。前人的研究表明，间充质干细胞能够改善肝纤维化，但hAMSCs治疗肝纤维化的潜能及其分子机制尚不清楚。. 本项目将重点研究hAMSCs在肝纤维化小鼠体内对受损肝脏组织的趋向性、并探讨hAMSCs治疗肝纤维化的潜能及分子机制。通过研究，我们发现hAMSCs移植进入肝纤维化小鼠体内后，部分hAMSCs可特异性迁移至受损肝脏组织、并通过抑制肝星状细胞（HSCs）的激活来改善肝纤维化小鼠肝脏功能、减少小鼠肝纤维化面积。体外，hAMSCs及其条件培养基（hAMSC-CM）可显著抑制肝星状细胞LX-2的激活，减少其胶原沉积。但hAMSCs共培养对HSCs的增殖和凋亡无明显影响。抗体芯片检测表明，hAMSCs可分泌大量IGFBP-3, DKK-3及DKK-1，通过抑制Wnt/β-catenin信号通路来抑制HSCs激活。本项目的实施可为肝纤维化的治疗提供一种可选方案，对进一步研究肝纤维化发病的分子机制，设计合理的治疗药物具有重要意义。.