肝脏前体细胞通过间质-上皮转化(MET)抑制星状细胞活化的作用及机制

In chronic injured liver, a large number of hepatic stellate cells (HSC) are activated, while hepatic progenitor cells (HPC) are seen periportally adjacent to HSC. Our previous studies found that co-culture of HPCs and activated HSCs led to a significant inhibition of HSC activation and proliferation. Those effects could be enhanced by the reversion of HPC epithelial-mesenchymal transition (EMT), which is named as mesenchymal-epithelial transition (MET), in addition to apoptosis of activated HSC. Accordingly, we hypothesized that HPC could modulate the activation of HSC through MET transition. .In this study we will first use the in vitro co-culture system of primary HPC and HSC. Comparing with HPC undergoing proliferation, differentiation and EMT by different growth factors, chemical substance or transforming growth factor-β (TGF-β), MET of HPC will be induced by TGF-β on-off withdraw (spontanous EMT turnover). HSC activation, proliferation and apoptosis, as well as extracellular matrix expression will be detected. Second, we will detect the three main MET signals Wnt/?-catenin，Notch and Hedgehog, and cytokines in the supernatant to explore the mechnisams and signaling of the MET effects on HSC. Third, to expend the understanding of the influence of HPC on HSC in vitro, we will use carbon tetrachloride (CCl4)induced liver fibrosis and regression rat model. By obseving HPC spontanous MET and BMP-7 induced MET, HSC activation and fibrosis marker will be evaluated. Finally, by cellular tracing assay we will doule label MET transited HPC with green fluorescence protein and E-cadherin promoter driven lacZ in vitro, and inject into fibrosis rat via portal vein. Histology, biochemical parameters of fibrogenesis and fibrolysis, most importantly the location and interaction of HPC on HSC by multi-fluoresence stain through confocal microscope, will be detected and evaluated. This study will help to understand the function of HPCs and their MET transition in liver fibrosis.

慢性肝损伤纤维化时前体细胞活跃。我们的前期研究发现病人纤维化减轻时，肝脏前体细胞常与星状细胞伴行并抑制其活化增生。这种作用与前体细胞发生的上皮－间质转化(EMT)后的逆转，即间质－上皮转化(MET)有关，但机制尚不清楚。本课题首先利用体外前体细胞和星状细胞共培养体系，观察前体细胞MET转化对星状细胞活化、凋亡和对细胞外基质的影响；第二通过检测前体细胞MET的三种主要信号通路 Wnt/?-catenin，Notch和Hedgehog，及上清液中细胞因子的变化探讨作用机制；第三利用四氯化碳大鼠纤维化及自发逆转模型，多标记荧光和组化染色分别观察前体细胞自发的MET及BMP-7诱导后的MET对星状细胞活化的影响；第四用细胞示踪技术将GFP和E-cad启动子/lacZ双重标记MET转化后的前体细胞回输纤维化大鼠体内，观察对星状细胞及纤维化消退的影响。本研究将为探讨纤维化中前体细胞的作用提供实验依据。

慢性肝损伤时，肝星状细胞（HSCs）被激活的同时，肝脏前体细胞（HPCs）也被激活，并参与损伤的修复。为明确肝脏前体细胞在慢性肝损伤时对星状细胞的作用，本研究首先利用transwell体外间接共培养体系，发现肝脏前体细胞不仅促进HSCs死亡的发生，而且可以促进HSCs发生凋亡，凋亡细胞的数量随着HPCs和HSCs比例的增加而增加。同时通过对纤维化相关基因的检测发现，肝脏前体细胞能明显抑制星状细胞活化，减少基质金属蛋白酶抑制剂-1（TIMP-1）和I型胶原（collagen I）的表达，并且抑制程度随着前体细胞和星状细胞比例的增加而增加。其次，我们采用经脾脏移植的方式，观察肝脏前体细胞对纤维化大鼠的影响。与假手术组相比，移植前体细胞后纤维化程度减轻、肝纤维化相关指标（α-SMA和TIMP-1）减少、collagen I分泌减少，并且肝功能（ALT和白蛋白）得到改善。以上结果表明，前体细胞不仅能够减轻肝脏纤维化程度，而且能够改善肝功能。再次，利用TGFβ1刺激前体细胞，模拟慢性肝损伤时的病理环境，观察前体细胞对星状细胞的作用。经TGFβ1（10ng/ml）刺激48小时的前体细胞表达间质细胞的标记物（α-SMA和TIMP-1）明显升高，干细胞标记物（白蛋白，甲胎蛋白）明显减少。发现TGFβ1刺激时间不同，对星状细胞的作用也不同。最后，通过PCR Array高通量的方式筛选，发现TGFβ1刺激12小时，SERPINE1在诱导肝脏前体细胞发挥抑制星状细胞活化方面起到重要作用。..在本研究中，我们通过体外间接共培养体系发现，前体细胞通过旁分泌作用可以直接抑制星状细胞活化、促进星状细胞凋亡并减少细胞外基质的表达。通过前体细胞移植入四氯化碳大鼠体内，提示前体细胞具有抑制纤维化、改善肝功能的作用。通过模拟慢性肝损伤时的病理环境，明确并优化前体细胞用于治疗慢性肝病的适宜时间及条件，为前体细胞治疗纤维化提供了一定的实验室基础。