胆汁酸对胰腺腺泡细胞内miR-155表达的影响及其机制研究

Severe acute pancreatitis (SAP) is a clinically severe disease, the most serious complication of which is systemic inflammatory response syndrome (SIRS); its mechanism is not fully understood. Toll-like receptors (TLRs) can cause inflammation or regulate the reaction by identifying endogenous substances and signal transduction. miR-155 is a newly discovered regulator of TLR signaling pathway, and its expression is regulated by PI3K/AKT signaling pathway, but it has not been reported that whether bile acids can activate the PI3K/AKT signaling pathway in pancreatic acinar cells. In our previous study, we found that trypsinogen activation can be detected in rat AR42J pancreatic acinar cells in 20 minutes after treated with bile acids (taurolithocholic acid 3-sulfate, TLC-S); meanwhile, the expression levels of multiple signal molecules in TLR signaling pathway were significantly increased. In view of the close relationship between miR-155 and TLR pathway, we hypothesize that bile acids can prompt the activation of TLR signaling pathway by PI3K/AKT signaling pathway mediated inhibition of miR-155. To test this hypothesis, we will establish severe pancreatitis model using pancreatic acinar AR42J cells and rat, employing techniques, such as real time PCR, Western blot, adenovirus vector transfection, to explore the role of miR-155 on the regulation of inflammation in severe pancreatitis and identify that bile acids can regulate the expression of miR-155 by PI3K/AKT signaling pathway. This study will provide a new target that can regulate inflammatory response in biliary acute pancreatitis and provide a new method for the treatment of pancreatitis.

重症急性胰腺炎诱发全身炎症反应综合征的机制尚未完全阐明。Toll样受体可通过识别机体内源性物质及信号传导引发炎症反应或对该反应进行调控。miR-155是一种新发现的TLR信号通路的调控因子，其表达受PI3K/AKT信号通路的调节，但胆汁酸能否激活胰腺腺泡细胞内PI3K/AKT信号通路尚未见报道。我们的前期实验发现，胆汁酸作用于大鼠胰腺腺泡AR42J细胞20分钟，既可在细胞内检测到TLR信号通路的多个信号分子表达水平明显升高。我们假设，胆汁酸通过PI3K/AKT信号通路抑制miR-155的表达促使TLR信号通路的激活。为验证这一假说，我们利用AR42J细胞和大鼠建立胰腺炎模型，采用RT-PCR、腺病毒载体转染等手段，从细胞及组织等多方面探讨miR-155对重症胰腺炎炎症反应的调控作用，明确胆汁酸调节miR-155表达的机制。以期发现调控胆源性胰腺炎时炎症反应的新靶点，为胰腺炎的治疗提供新方法

急性胰腺炎（AP）是临床上常见的一种急腹症，80%病因是由于胆囊结石和过量饮酒引起。 急性胰腺炎的病理生理学通常分为三个阶段：第一阶段，胰腺腺泡细胞中胰蛋白酶的过早激活，第二阶段，胰腺自身的炎症，第三阶段，胰腺外器官炎症。胰蛋白酶原在胰腺腺泡细胞内异位激活是AP的始动因素，腺泡细胞的病变在胰腺炎的发生发展中处于核心地位。本项目以胰蛋白酶原的细胞内激活为靶点，探讨急性胰腺炎发生发展的潜在机制。第一部分，课题组通过细胞实验探索了胆汁酸对胰腺腺泡细胞内对miR-155 表达的影响，以及胆汁酸是否通过AKT信号通路来影响miR-155的表达，结果未发现两者之间存在明显相关性。第二部分，采用生物信息学方法，构建了AP发生发展相关的ceRNA网络。使用差异表达分析方法筛选AR42J细胞的miRNA微阵列中差异表达前10位miRNA.，使用miRWalk和miRDB网站预测可能与这前10位miRNA存在ceRNA关系的lncRNA、mRNA，使用cytoscape软件构建ceRNA网络，并预测可能影响AP的lncRNA，mRNA，miRNA。我们构建的ceRNA网络包括16个lncRNA，9个mRNA和2个miRNA。通过GO功能富集分析、KEGG通路富集分析，富集到的功能和通路包括离子通道活动、主动跨膜转运蛋白活性， JNK / MAPK、 PI3K / Akt信号传导通路。第三部分，研究了膜联蛋白ANXA2 和 lncRNA Fendrr的相互作用对胰腺腺泡细胞凋亡的影响，结果发现，在雨蛙素诱导的胰腺炎细胞模型中，ANXA2表达增高， 具有促进胰腺细胞凋亡的作用，而LncRNA Fendrr同样表达增高，而 ANXA2通过嵌合LncRNA Fendrr发挥其凋亡促进作用。该项目的研究为进一步理解急性胰腺炎的发生机制提供了新的思路，并为急性胰腺炎的治疗提供了潜在的治疗靶点。

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