FoxA2通过阻断和逆转肝脏前体细胞EMT而抑制肝纤维化进展的机制研究

As progeny of hepatic stem cells, hepatic progenitor cells emerge and expand in periportal areas of the injured liver when the regenerative capability of hepatocytes and cholangiocytes are impaired, thus serving as the major cells for liver injury repair and tissue regeneration. Previous studies show that TGF-β1, the major fibrogenic factor in the liver, may induce hepatic progenitor cells to display fibroblastic phenotype through epithelial-mesenchymal transition (EMT), and chronic and constant treatment of TGF-β1 further makes them obtaining tumor initiating cell properties. We find that epithelial growth factor (EGF) appears to be the specific cytokine with an opposite function to TGF-β1 allowing for suppressing and reversing EMT of hepatic progenitors through phosphorylation of ERK1/2 and Akt. Furthermore, the expression of Forkhead Box Protein A2 (FoxA2) reduced significantly in TGF-β1-induced EMT process and recovered in EGF-regulated EMT-reversing process. Therefore, we hypothesize FoxA2 might be the major transcription factor involving the EMT blocking and reversing process of hepatic progenitors, which may slow down the progression of liver fibrosis. In this study, We are going to find the mechanism of FoxA2 on EMT transition and liver fibrogenesis of hepatic progenitors from the following three aspects. First, we would like to confirm the signal pathway of EGF on the transcription, expression and function of FoxA2 by EGF receptor inhibitor, ERK1/2 inhibitor and Akt inhibitor. Secondly, we are going to use FoxA2 over-expressing and FoxA2 siRNA hepatic progenitors to find the blocking and reversing effects of FoxA2 on EMT transition of hepatic progenitors and the interactions of FoxA2 with the gene promoters and the transcription factors involved in EMT transition, cell proliferation and differentiation. Finally, we will transplant FoxA2 over-expressing and FoxA2 siRNA hepatic progenitors into rat, and induce liver fibrosis by CCl4 and fibrosis regression by withdraw CCl4 in order to find the contribution of hepatic progenitors with different FoxA2 expressing status to liver fibrosis. We hope this study could provide evidence for understanding the behavior of hepatic progenitors in the progression and regression of liver fibrosis and reversion of liver fibrosis.

肝脏前体细胞是肝脏损伤修复的关键细胞，但转化生长因子(TGF-b1)会使其发生上皮-间质转化(EMT)成为肌成纤维细胞。我们的研究发现表皮细胞生长因子(EGF)可通过ERK1/2和Akt途径阻断和逆转前体细胞的EMT，且叉头蛋白FoxA2是此过程中变化显著的转录因子，推测FoxA2是阻断和逆转肝脏前体细胞EMT抑制肝纤维化的关键因子。本研究首先利用EGF通路抑制剂探讨EGF阻断和逆转前体细胞EMT过程中调控FoxA2表达与功能的信号分子；然后采用过表达和抑制FoxA2表达的前体细胞检测FoxA2与EMT、增殖分化基因和转录因子的相互作用，明确FoxA2干预前体细胞EMT转变的机制；最后将过表达和抑制FoxA2表达的前体细胞移植到大鼠中，并制备四氯化碳肝纤维化与纤维消解模型，探讨干预前体细胞的FoxA2对纤维化进展与消解的影响。期待为阐明肝脏前体细胞在纤维化进展和消解中的作用提供依据。

肝纤维化是多种病因所致的慢性肝病损伤修复与疾病进展的重要病理过程，在成熟肝细胞增殖被抑制的情况下肝脏前体细胞是肝脏修复再生的关键细胞。FoxA2作为肝脏前体细胞高表达的转录因子，在致纤维化因子TGF-b1诱导的EMT转变过程中表达显著下降。 EGF不仅通过EGFR/Akt信号通路拮抗TGF-b1抑制FoxA2表达的作用，还可通过EGFR/ERK1/2/Akt信号通路增加EMT细胞FoxA2表达。在此EMT/MET转变过程中FoxA2与辅因子Npm1和HSP90的结合呈先升高后降低的变化趋势。鉴于TGF-b1是一种关键的致纤维化因子，而且体外EGF具有拮抗TGF-b1的作用，我们应用EGF与EGF抗体处理BDL小鼠，发现EGF抗纤维化效果不明显，而EGF抗体能够通过抑制肌成纤维细胞增殖而减轻BDL小鼠肝纤维化。.敲减FoxA2的实验结果显示FoxA2具有抑制肝脏前体细胞增殖、促进其朝肝细胞方向分化的作用。尽管肝脏前体细胞高表达FoxA2但HNF4a表达水平很低，为对比肝脏前体细胞移植与成熟肝细胞细胞移植治疗肝损伤的机制与效果，本研究将肝脏前体细胞、过表达HNF4a的肝脏前体细胞和抑制HNF4a表达的肝脏前体细胞分别移植到CCl4损伤的NOD/SCID小鼠中，发现抑制HNF4a表达后，肝脏前体细胞无法定位于肝脏，而肝脏前体细胞与过表达HNF4a的肝脏前体细胞可以整合到损伤的肝组织中，且过表达HNF4a的肝脏前体细胞较对照肝脏前体细胞迁移能力更低、组织纤维化程度更低，表明过表达HNF4a的肝脏前体细胞是更好的细胞移植治疗肝脏疾病的备选细胞。