肝星状细胞自噬的“选靶”差异对实验性肝纤维化治疗的影响及钙调机制研究

For the hepatic fibrosis, any therapeutic approach should be applied to inhibit or promote the autophagy in hepatic stellate cell (HSC)? Given our previous study with a focus on basic role of autophagy in the process of hepatic fibrosis, and the revealing of critical impact of calcium mobilization on cellular differentiation as well as selective gene silencing, it is raised a brand new hypothesis that the selective autophagy in HSC might be mediated by intracellular calcium messenger-dependent calcium mobilization, resulting in different outcomes for hepatic fibrosis therapy. Therefore, this study firstly aims to characterize the selective autophagy associated morphologic and molecular biologic aspects. Secondly, fluorescent imaging-live cell workstation will be used to record the regulatory pattern of different calcium second messenger-indued calcium flux, and further to investigate the relationship between calcium mobilization and selective autophagy in HSC. Finally, the invo and in vitro effect of modified selective autophagy in HSC will be determined on experimental hepatic fibrosis in the present of different medicines. Taken together, our proposal will offer a new insight into the role of HSC autophagy in hepatic fibrotic treatment, and will reveal a novel mechanism responsible for calcium-regulated selective autophagy in this cell. Moreover, it will provide reliable scientific evidence for screening and evaluating novel agents for anti-hepatic fibrosis in the future.

对于肝纤维化治疗，应该采取抑制自噬？还是促进自噬？基于前期关于肝星状细胞（HSC）自噬在肝纤维化进程中一般性作用的研究，结合钙动员可影响细胞分化、决定基因选择性沉默的发现，我们提出了大胆的新设想：细胞内钙信使依赖性钙动员可能介导HSC自噬的"选靶"差异,并因此对肝纤维化治疗产生不同影响。为此，本项目计划首先从形态学、分子生物学水平考证HSC客观存在不同底物形式选择性自噬及表现特征；通过体外干预，利用高速荧光影像－活细胞工作站观察不同第二钙信使对钙离子流的调节方式，进一步探讨HSC内钙动员与选择性自噬的关系；最后，从离体和整体水平研究选择性调节HSC自噬状态对肝纤维化实验治疗的影响。这将有助于通过揭示HSC发生选择性自噬的钙调控新机制，深化对自噬在肝纤维化治疗作用的认识，为将来基于自噬理论遴选、评价抗肝纤维化新药物提供必要的科学依据。

肝硬化是临床常见病，肝纤维化是肝硬化的必经病理过程。在既往关于细胞自噬（Autophagy）与肝纤维化关系的研究基础上，我们进一步提出肝纤维化的发生和治疗可能与自噬的选择性相关，我们在国家自然科学基金的资助下，利用多种影像学技术、基因检测和蛋白质分析等手段，首先描述了肝纤维化关键细胞—肝星状细胞（HSC）发生线粒体自噬、内质网自噬、脂滴自噬三种选择性自噬的影像及分子表现特征；其次，揭示了HSC通过内质网自噬促进肝纤维化进程的初步规律。抑制P-eIF2a介导的内质网自噬可促进HSC凋亡，抑制细胞活化，减少胶原纤维的生成；第三，初步发现PDGF、TGFb1可诱导TPC2蛋白下调，从而触发NAADP介导的细胞钙动员。第四，发现熊去氧胆酸（UDCA）可抑制HSC活化过程中线粒体蛋白NIX/BNIP3L的表达并减少胶原沉积，干扰线粒体自噬发生是UDCA抗肝纤维化作用新机制。本研究客观阐述了选择性HSC自噬与肝纤维化的复杂关系，指出在制定肝纤维化治疗策略时，并非简单地促进或抑制非特异性自噬，应结合特定药物、特定选择性自噬状态综合考虑。总之，本项目丰富了肝纤维化机制的理论认识，实验数据对推动UDCA治疗肝纤维化临床研究提供了可靠的参考依据。本项目已培养毕业研究生2人，在培养3人，发表各种形式论文12篇。参加国际、国内学术交流4次。