钠钙交换体NCX1介导钙超载在急性胰腺炎发生发展中的作用及其机制研究

Previous studies have shown that calcium overload in acinar cell are the early steps and contributing factors in pathogenesis of pancreatitis, and the degree of calcium overload was positively correlated with the severity of acute pancreatitis. Our results of previous experiments have shown that sodium calcium exchanger1(NCX1), as the important membrane ion channel, not only mediated calcium overload in acinar cells, but also highly expressed in early pancreatitis. However, the regulation mechanism that NCX1 mediated calcium overload to promote pancreatitis is still not clear. The dual luciferase experiments showed that the activity of NCX1 promoter can be significantly up-regulated when pancreatitis was induced by caerulein. We found that nuclear transcription factor NF-κB, as the important downstream regulatory molecules of calcium overload, had several binding sites on the promoter of NCX1. It demonstrates that NCX1 may promote the activation of NF-κB and further up-regulated the transcriptional expression of NCX1, which form a positive feedback pathway to promote the progress of acute pancreatitis. Therefore, NCX1 is likely to be an important target for the treatment of acute pancreatitis. Based on above hypothesis, this study will illustrate NCX1's important role in pancreatitis through molecular and functional mechanism using laser confocal, animal model building, EMSA and ChIP, which will provide a new theoretical basis for intervening pancreatitis through targeting NCX1.

既往研究表明腺泡细胞钙超载是胰腺炎发病的早期环节及加重因素，钙超载的程度与胰腺的病理变化及病程进展呈正相关。我们前期的预实验发现钠钙交换体NCX1作为细胞膜上重要的离子通道不仅介导了胰腺炎的钙超载，还在胰腺炎发生早期表达明显上调，但NCX1通过钙超载促进胰腺炎的调控机制并不清楚。双荧光素酶实验发现雨蛙素体外诱导胰腺炎后可以明显上调NCX1的启动子活性，而NF-KB作为胰腺炎钙超载下游重要的调控分子，生物信息学预测发现其在NCX1启动子区域有结合位点，提示NCX1介导的钙超载可能通过促进NF-ΚB的活化上调NCX1的转录表达，从而形成一种正向反馈调控通路促进急性胰腺炎的进展，因此NCX1很可能成为治疗胰腺炎的重要靶点。本课题拟采用激光共聚焦，动物模型构建以及EMSA和ChIP等分子生物学技术，从分子及功能机制阐明NCX1在胰腺炎中的重要作用，为以NCX1为靶点的胰腺炎防治提供新的理论依据

急性胰腺炎（Acute Pancreatitis AP）是多种病因导致胰酶在胰腺内被激活后引起的炎症反应，起病急，预后差，临床死亡进率近30%，因此急性胰腺炎的治疗多年来一直是人们研究的热点。有研究表明胰腺腺泡细胞钙超载是导致胰腺炎发生、促进胰腺炎发展的重要机制，而细胞膜钙通道蛋白介导的外钙内流是导致钙超载的主要原因。钠钙交换体NCX1蛋白可介导细胞内外的Na+、Ca2+离子交换，本课题从分子研究及功能测定等多方面获得NCX1参与急性胰腺炎发生发展的相关证据，揭示其参与胰腺炎调控的机制是通过介导钙超载激活核转录因子NF-ΚB来上调NCX1的表达，从而形成一条正反馈调控通路，引发胰腺炎的炎性反应介质的级联反应，造成急性胰腺炎的迅速恶化。.课题首先证实在胰腺腺泡细胞AR42J中存在NCX1蛋白表达，随后利用雨蛙素处理胰腺腺泡细胞AR42J建立了胰腺炎细胞模型，通过Western Blot实验检测发现随雨蛙素处理时间延长，NCX1表达随之增加，提示NCX1在雨蛙素造成胰腺炎中可能具有重要作用；分析NCX1的启动子区后发现含有1个NF-κB以及3个SP1的结合位点，利用双荧光素酶报告载体证实NF-κB及SP1能够上调NCX1的启动子活性，并且在雨蛙素造成的胰腺炎模型中这两种转录因子的表达都有不同程度上调，提示胰腺炎过程中转录因子NF-κB及SP1表达升高，并上调了NCX1蛋白表达；进一步的钙测定实验发现雨蛙素处理后细胞内Ca2+增加，而抑制NCX1表达的AR42J细胞内Ca2+无明显增加，说明NCX1确实介导了外钙内流造成胰腺炎钙超载。同时，课题组利用雨蛙素腹腔注射大鼠成功建立体内胰腺炎模型，并在组织标本中通过免疫组化和HEA染色进一步证实了胰腺炎发展过程中NF-κB及SP1上调NCX1表达的作用。本研究不仅是对胰腺炎的发病机制很好的补充，也为胰腺炎的防治提供了新的作用靶点。