外泌体通过递送miR-130a介导胰腺腺泡细胞-星状细胞间通讯参与胰腺纤维化分子调控

Pancreatic fibrosis, the main pathological features of chronic pancreatitis (CP), is closely related to the activation of the pancreatitic stellate cell (PSC). But its regulatory mechanism still needs to be further researched. Based on our early experiments about miRNA chip, mRNA chip and bioinformatics analysis, we prompt a hypothesis that “During CP the pancreatic acinar cells can release exosomes in which miR-130a was carried. The carried miR-130a then was delivered to the recipient cell (PSC) and activate PSC targeting PPARγ, finally promote pancreatic fibrosis” . In order to verify the truth of this hypothesis, a tracer technique of fluorescence labelling exosome and the carried RNAs was using to demonstrate the transportation of the exosome and the carried RNAs; in vitro and in vivo study of “gain of function” or “loss of function” was using to demonstrate effects of miR-130a on PSC activation and pancreatic fibrosis; luciferase experiment and RIP (RNA Binding Protein Immunoprecipitation) was using to demonstrate whether the carried miR-130a were combined to it’s target gene -PPARγ.Thereby to demonstrate the function mechanism of communications between pancreatic acinar cells and PSC mediated by exosome and the carried miR-130a. The success of this study will undoubtedly provide solid theoretical basis for the studies of pathogenesis, prevention and treatment of CP.

胰腺纤维化是慢性胰腺炎（CP）的主要病理特征，与胰腺星状细胞（PSC）的活化密切相关，但其调节机制仍有待深入研究。本课题组基于前期miRNA芯片、mRNA芯片筛选及生物信息学分析结果提出假说：“被激活的胰腺腺泡细胞释放外泌体，外泌体携载miR-130a， miR-130a递送至受体细胞（PSC）后结合并抑制其靶基因（PPARγ），从而促进PSC活化及胰腺纤维化”。 为验证这一假说，本研究拟通过荧光标记并追踪外泌体及其携载的RNAs在细胞间的转运；通过体外和体内功能获得性研究及功能缺失性研究验证miR-130a对PSC活化及胰腺纤维化等表型的影响；通过双荧光素酶报告实验及RNA结合蛋白免疫沉淀实验(RIP)验证miR-130a是否与PPARγ直接结合。从而阐述外泌体递送的miR-130a介导胰腺腺泡细胞-PSC间通讯的作用机制。本课题的完成，将为CP的发病机制及防治研究提供新的线索。

外泌体作为内源性miRNA转运系统参与多种病理生理过程。然而其在胰腺纤维化中的功能和机制依旧未知。有效控制胰腺星形细胞（PSC）的激活对治疗胰腺纤维化至关重要。本研究利用外泌体的荧光标记及动态示踪检测证明PSC能够摄取AR42J分泌的外泌体。RNA芯片检测TLC处理前后AR42J细胞差异表达的外泌体miRNA，并与PSC激活后差异表达基因进行共表达分析，预测miR-130a/PPAR-γ关系对。RT-PCR证实miR-130a在外泌体中富集。在胰腺炎细胞模型中利用Western Blot证实miR-130a具有促进PSC的激活、胶原纤维合成并抑制PPAR-γ表达的作用。动物模型中miR-130a inhibitor具有抑制PSC的激活、胶原纤维合成的作用，还能够保护内皮细胞功能和胰腺内分泌与外分泌功能。荧光素酶标记实验证实PSC内miR-130a与PPAR-γ的 3’UTR存在直接结合的关系。利用miRGen预测miR-130a的转录因子为RREB1。因此我们认为受损的胰腺腺泡细胞（AR42J）可能通过PDGF/Ras/RREB1通路分泌外泌体miR-130a并通过抑制PPAR-γ促进PSC活化及胰腺纤维化。本研究揭示了外泌体miR-130a参与激活PSC的机制，为抑制胰腺纤维化提供可能的治疗靶点。