Hippo通路关键分子Mst1在慢性胰腺炎纤维化中的作用机制研究

The fibrosis of pancreatic parenchyma is the essential pathological change of chronic pancreatitis (CP), and also the potential pathway for targeted therapy of CP.Pancreatic stellate cell (PSC) is recognized as the critical cell in the pathogenesis of pancreatic fibrosis and primarily activated by transformation growth factor beta 1 (TGF-β1). Hippo pathway, with mammlian Ste-20 like kinase 1(Mst1) as one of its core kinases, has recently been found to be a significant signaling pathway that balances the proliferation and apoptosis of cells in different organs and the crosstalk between Hippo and TGF-β1 is confirmed in the regulation of proliferation and fibrosis. However, the role of Hippo pathway in the pathogenesis of CP is still unclear.In the preliminary studies, we found that the level of Yes-associated protein (YAP), an important transcription coactivator in the downstream of Mst1, was significantly increased in the tissue of CP patients. Moreover, we built CP rat model induced by dibutyltindichloride (DBTC) infusion into the tail vein and found that pancreatic Mst1 level was paralleled with the extent of fibrosis of the pancreatic tissue. Thus, we assumed that Hippo pathway plays important role in the fibrosis of CP. We will firstly continue to explore the association between Mst1 and TGF-β1, PSC state, and pancreatic fibrosis in CP and normal specimen both from human surgery specimen and rat model. Second, we will analysis the differential expression of Hippo and TGF-β pathway genes in PSCs in quiescent and activated state. Third, we will build Mst1 and Mst1 shRNA lentivirus vector. At last, we will investigate the regulation of Mst1 upon PSC activation in vitro and upon PSC activation and pancreatic fibrosis in vivo with transfection of lentivirus vectors.

胰腺纤维化是慢性胰腺炎(CP)的主要病理表现。胰腺星状细胞(PSC)在胰腺纤维化中发挥重要作用，转化生长因子-β1(TGF-β1)是其最主要的活化因子之一。最近研究发现，Hippo通路关键分子Mst1对组织修复发挥重要作用，且Hippo通路与TGF-β通路存在串话，参与调控细胞增殖和组织纤维化。目前Hippo通路在CP纤维化中的作用尚不明确。我们前期在人CP组织中发现Mst1下游分子YAP水平显著上调，在CP大鼠模型中发现Mst1水平与纤维化进程一致。本课题拟进一步在人胰腺手术标本、大鼠胰腺组织、离体的静息/活化PSC三个水平证实Mst1与TGF-β1下游分子、PSC状态及胰腺纤维化的相关性；构建Mst1和Mst1 shRNA慢病毒载体，在离体PSC中研究Mst1对其活化的调控，在动物模型中研究Mst1对PSC活化及胰腺纤维化的作用。进一步阐明胰腺纤维化机制，为抑制胰腺纤维化提供治疗靶点。

胰腺星状细胞（PSC）活化是胰腺纤维化核心机制，阐明促进和维持PSC活化的信号通路有助于阐释慢性胰腺炎（CP）发病机制。由于长期以上缺少细胞系限制其分子机制的研究，本研究通过采用逆转录病毒载体的方法将SV40大T抗原（SV40 LT）和人端粒酶逆转录酶（hTERT）导入到原代PSC中，建立永生化PSC细胞系。通过一系列的验证实验表明该永生化细胞系可替代原代细胞的分离和培养。Hippo信号通路被报道参与其它组织纤维化过程，但在胰腺中尚未报道。本研究在永生化PSC中构建了Mst1过表达和敲减的细胞株，建立了永生化细胞系病毒转染的技术体系。我们的结果显示Hippo信号通路关键性蛋白Mst11/2和磷酸化的YAP在CP中表达量明显发生改变。过表达或敲减Mst1可通过改变部分TGF-β下游基因的表达，参与细胞外基质的形成；此外细胞实验结果显示过表达Mst1可缓解TGF-β诱导的PSC活化。最后通过在DBTC诱导的CP大鼠模型中，通过Mst1过表达腺病毒载体进行干预治疗，结果显示干预Mst1表达可缓解CP大鼠的纤维化程度。本研究结果有助于深入的认识慢性胰腺炎的机制，并初步验证干预Mst1可缓解纤维化，为临床诊断治疗指示新的方向。