Caspase/Hedgehog关联的凋亡诱导代偿性增殖在肝再生中的作用研究

Liver regeneration is an unique protective response to diverse forms of injury which can be affected by many liver diseases. In clinical settings, the feasibility and prognosis of any treatment for liver diseases are largely dependent on the regenerative efficacy of remnant liver. Cell apoptosis and cell proliferation,two basic bological process, have been found closely linked as apoptosis-induced compensatory proliferation which paves a new way for understanding of important physiological and pathological conditions.In this project, the classical animal model for liver regeneration based on partial hepatectomy will be used to observe the temporal and spatial correlations between caspase activity and the expression of several components(Hedgehog ligands,Ptc,Smo and Gli) in Hedgehog signaling pathway, also known as morphogen, during liver regeneration process. Then, we will carry out in vitro experiments using mixed and transwell co-culture systems to examine whether dying cells could stimulate the growth of living hepatocytes and subsequently to identify molecules mediating caspase/hedgehog communication. And the last part of this project is to investigate the effects of specific inhibitors or agonists for caspases and hedgehog signaling pathways on liver function, liver regeneration and survival rate of mice performed with massive hepatectomy. The prospective results will help unravel the detailed procedures and key molecules mediating the crosstalk between cells and among different signaling pathways,to better understand diseases characterized by functional deficiency or cell overgrowth, and to design new methods to enhance liver regeneration and repair.

肝再生是机体的一种独特修复反应，许多肝胆疾病及其处理影响了该反应的有效发挥，临床上能否实施治疗和预后如何很大程度上取决于肝脏的再生修复能力。细胞凋亡和细胞增殖两种普遍生命现象间的关联即凋亡诱导代偿性增殖为研究重要生理过程和疾病状态提供了全新视角。本项目首先在小鼠肝部分切除后肝再生模型上动态观察肝再生过程中细胞凋亡关键蛋白酶caspase活性与肝脏形态发生信号通路Hedgehog(Hh)关键成分Hh配体、Ptc、Smo、Gli等表达间的时相和定位关系，其次通过混合和非接触式共培养探究caspase/Hh通路间的作用方式和关联分子，在此基础上利用抑制剂和激动剂特异性调节上述两条途径活性，观察对扩大肝切除小鼠肝脏再生和术后存活的影响。细胞凋亡与增殖的关联研究有助于揭示细胞之间、信号通路之间复杂网络联系，深化对功能细胞不足和细胞增殖过度为特征的疾病的认识，并以此为线索寻找增强肝脏再生反应的有效手段

鉴于caspase介导的凋亡诱导代偿性增殖现象尚未在培养人肝细胞和肝损伤病理过程中得到证实，本课题首先通过基因转染成功获得稳定表达黄色荧光蛋白Venus的人肝细胞L-02（L-02/Venus)，在此基础上用不同强度X光辐射诱导L-02/Wt细胞凋亡，确定12Gy为适宜辐照剂量；建立诱导凋亡L-02与L-02/Venus的共培养体系，初步掌握后者增殖状态以及影响因素。在大鼠90%门静脉结扎和联合相同范围的胆管结扎诱导的肝萎缩-肥大综合征(损伤和增殖共存）模型上，用生化和组织学方法均检测到凋亡关键蛋白caspase-3的活化，并且与细胞增殖标志蛋白Ki-67表达共存，萎缩肝上还呈现凋亡与增殖在程度和时相上的伴随关系，而肥大肝脏也有caspase-3活性表达，为凋亡诱导代偿性增殖存在于肝脏损伤过程提供了支持证据，此现象国内外未见报道。上述研究结果有助于后续开展凋亡诱导代偿性细胞增殖分子机制及其病理生理意义的研究。