Apoptosis and Necrosis in Pancreatitis

描述（由申请人提供）：炎症和实质组织损伤是胰腺炎的标志。特别是，严重的坏死是该疾病的主要并发症。在过去的十年中，在了解胰腺炎炎症反应的机制方面取得了重大进展。相比之下，我们对胰腺腺泡细胞死亡的机制知之甚少。坏死的机制在很大程度上是未知的。确定了介导细胞凋亡的关键信号；然而，它们在疾病过程中的作用仍然不清楚，并且尚未在胰腺炎中进行研究。细胞死亡途径在病理性胰蛋白酶激活中的作用，胰蛋白酶是胰腺炎组织损伤的重要标志，尚未被探索。我们的初步数据表明，在胰腺炎实验模型和受胆囊收缩素（CCK）刺激的胰腺腺泡细胞中，关键的坏死和凋亡机制：聚（adp -核糖）聚合酶（PARP）、线粒体功能障碍、半胱天冬酶（特异性半胱氨酸蛋白酶）和转录因子NFkappaB被激活。对于目前的应用，我们假设在胰腺炎中，坏死和凋亡信号通路是相互关联的。PARP的激活和线粒体失能导致ATP耗竭和坏死。另一方面，效应半胱天冬酶通过使PARP和胰蛋白酶失活介导细胞凋亡和限制坏死。NFkappaB负调控效应caspases，从而在胰腺炎中发挥抗凋亡作用。因此，PARP、线粒体功能障碍、caspase和NFkappaB在决定细胞凋亡与坏死型腺泡细胞死亡和胰腺炎严重程度之间的平衡中起着核心作用。我们提出以下具体目标为本应用程序：1)。确定PARP在实验性胰腺炎和体外CCK刺激胰腺腺泡坏死和凋亡中的作用。2）. 确定线粒体功能障碍在实验性胰腺炎和体外CCK刺激胰腺腺泡中坏死和凋亡的作用。3)确定caspase在实验性胰腺炎和体外CCK刺激胰腺腺泡中坏死、凋亡和胰蛋白酶激活中的作用。4)。测定NFkappaB在实验性胰腺炎和体外CCK刺激胰腺腺泡中坏死和凋亡的作用。实现这些目标的测量将包括胰腺炎的测量，细胞凋亡和坏死的形态学表征，胰腺内半胱天酶和胰蛋白酶的激活，细胞色素c释放，线粒体膜电位，ATP水平，以及使用Western blot和凝胶转移分析，酶和荧光分析的NFkappaB激活。这些实验的结果将描述急性胰腺炎中调节坏死和细胞凋亡的关键分子机制，这将导致新的治疗策略。

DESCRIPTION (provided by applicant): Inflammation and parenchymal tissue damage are hallmarks of pancreatitis. In particular, severe necrosis is a major complication of the disease. Over the past decade, significant progress has been achieved in understanding the mechanisms of the inflammatory response of pancreatitis. In contrast, very little is known about the mechanisms of pancreatic acinar cell death. Mechanisms of necrosis are largely unknown. Key signals mediating apoptosis have been established; however, their roles in disease processes remain obscure, and they have not been investigated in pancreatitis. The role of cell death pathways in pathologic trypsin activation, an important marker of tissue damage in pancreatitis, has not been explored. Our preliminary data indicate that key necrotic and apoptotic mechanisms: poly (ADP-ribose) polymerase (PARP), mitochondrial dysfunction, caspases (specific cysteine proteases), and the transcription factor NFkappaB are activated in experimental models of pancreatitis and in pancreatic acinar cells stimulated with cholecystokinin (CCK). For the present application, we hypothesize that in pancreatitis, necrotic and apoptotic signaling pathways are interrelated. Activation of PARP and mitochondrial de-energization leads to ATP depletion and necrosis. On the other hand, effector caspases mediate apoptosis and limit necrosis by inactivating PARP and trypsin. NFkappaB negatively regulates effector caspases and, thus plays an anti-apoptotic role in pancreatitis. Thus PARP, mitochondrial dysfunction, caspases, and NFkappaB play central roles in determining the balance between apoptotic versus necrotic type of acinar cell death and the severity of pancreatitis. We propose the following specific objectives for the present application: 1). Determine the role of PARP in necrosis and apoptosis in experimental pancreatitis and in vitro, in pancreatic acini stimulated with CCK. 2). Determine the role of mitochondrial dysfunction in necrosis and apoptosis in experimental pancreatitis and in vitro, in pancreatic acini stimulated with CCK. 3) Determine the role of caspases in necrosis, apoptosis, and trypsin activation in experimental pancreatitis and in vitro, in pancreatic acini stimulated with CCK. 4). Determine the role of NFkappaB in necrosis and apoptosis in experimental pancreatitis and in vitro, in pancreatic acini stimulated with CCK. Measurements to achieve these goals will include measures of pancreatitis, morphologic characterization of apoptosis and necrosis, intrapancreatic activation of caspases and trypsin, cytochrome c release, mitochondrial membrane potential, ATP levels, and NFkappaB activation by using Western blot and gel shift analyses, enzymatic and fluorimetric assays. The result of the experiments in the proposed specific objectives will be delineation of key molecular mechanisms regulating necrosis and apoptosis in acute pancreatitis, which will lead to novel therapeutic strategies to treat the disease.