Hippo通路介导的胞外囊泡促进胰腺星状细胞（PSC）活化的机制研究

The fibrosis of pancreatic parenchyma is the essential pathological change of chronic pancreatitis, and pancreatic stellate cell (PSC) is recognized as the critical cell in the pathogenesis of pancreatic fibrosis. With the support of young scientist fund of NSFC (81300355), we have identified the role of Hippo pathway (Mst1-YAP) in regulating PSC activation and pancreatic fibrosis. Recent studies suggested that the Hippo pathway may involve the regulation of the microenvironment during PSC activation, and extracellular vesicle (EV) is the potential pathway. The experiments we have finished showed that some exosomes RNAs are biomarkers of chronic pancreatitis, the EV derived from Mst1-knocked-out PSC can activate quiescent PSC, and Hippo can regulate the expression of CCN2 and miRNAs in EV. In this study, we aim to further verify that the Hippo pathway regulates the EV loading and the abundance of miRNA and CCN2 in the PSC. Then, we will analyze the mechanism of Hippo pathway improving the abundance of miRNA and CCN2 in PSC through mutation of YAP-related locus. Moreover, we will analyze the effect of miRNA and CCN2 in EV on resting PSC by ExoFectin method. We hope this study will elucidate preliminary the mechanism of Hippo-mediated EV pathway promoting the activation of PSC and provide a basis for future treatment.

胰腺纤维化是慢性胰腺炎主要病理表现，胰腺星状细胞(PSC)活化在其发病机制中发挥重要作用。申请人前期主持青年项目证实Hippo通路(Mst1-YAP)可调控PSC活化和胰腺纤维化。近期研究提示Hippo通路可能参与调控PSC活化微环境，胞外囊泡(EV)是其潜在途径。申请人预实验发现有外泌体RNA可作为慢性胰腺炎标志物，Mst1敲减PSC来源的EV可激活静息PSC，Hippo可影响PSC分泌EV的CCN2和miRNAs表达。本项目拟通过转基因及补偿实验进一步验证Hippo通路调节PSC的EV装载及miRNA和CCN2等的丰度，在PSC中突变YAP相关位点分析Hippo通路提高miRNA和CCN2丰度的机制，通过ExoFectin将miRNA或CCN2转入EV，分析EV中miRNA和CCN2对静息PSC的活化作用。希望初步阐明Hippo通路介导的EV促进PSC活化的机制，为今后治疗提供基础。

慢性胰腺炎（CP）是各种病因导致的胰腺组织进行性损伤，胰腺纤维化是CP最主要的病理学特征，也是引起胰腺功能障碍的重要原因，胰腺星状细胞（PSC）活化是胰腺纤维化的核心机制，阐明促进PSC活化的信号通路有助于进一步阐释CP发病机制。本研究旨在探索Hippo通路通过EV传递miRNAs和CCN2等信号参与PSC间细胞通讯的机制，评估EV载YAP抑制剂药物进行胰腺纤维化治疗的潜在价值。研究成功构建了大鼠PSC永生化细胞系，成功在PSC永生化细胞中构建了Mst1敲减和过表达的细胞株，通过miRNAs芯片检测发现miR-211、miR-29a、miR-130a和miR-625的表达与Mst1负相关，证实miR-211、miR-130a及CCN2三者与Hippo通路的活性明显相关，miR-145-5p通过作用于CCN2抑制HPSC活化、迁移和侵袭，以及EV分泌和功能。利用持续性激活的Yap1（S127A）以及Yap1（8SA）过表达腺病毒观察Hippo通路的持续性激活对HPSC增殖活化、EV分泌和微环境的影响。利用VGLL4以及缺失TDU结构域的VGLL4 (delTDU)过表达腺病毒，并感染HPSC，观察其抑制Hippo通路活性对HPSC增殖活化、EV分泌和微环境的影响。发现Dasatinib能通过抑制Hippo通路活性来抑制HPSC的增殖活化、胰腺纤维化和EV分泌。项目执行期间，申请者以第一（含共同）、共同通讯作者发表SCI论文共13篇，获批1项实用新型专利，入选上海市卫生系统优秀青年医学人才培养计划，获上海市科技进步一等奖（慢性胰腺炎临床诊治关键技术的研究与应用，第5，已公示）。