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，在了解胰腺炎的炎症反应机制方面取得了重大进展。相反，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。介导细胞凋亡的关键信号已经建立，然而，它们在疾病过程中的作用仍然不清楚，并且它们在胰腺炎中尚未被研究。病理性胰蛋白酶激活是胰腺炎组织损伤的重要标志，但细胞死亡途径在其中的作用尚未研究。我们的初步数据表明，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，半胱氨酸蛋白酶（特定的半胱氨酸蛋白酶），和转录因子NF κ B在胰腺炎的实验模型和胰腺腺泡细胞刺激胆囊收缩素（CCK）被激活。 对于本申请，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的激活和线粒体脱钙导致ATP耗竭和坏死。另一方面，效应半胱天冬酶通过灭活PARP和胰蛋白酶介导细胞凋亡并限制坏死。NF κ B负性调节效应半胱天冬酶，因此在胰腺炎中起抗凋亡作用。因此，PARP、线粒体功能障碍、半胱天冬酶和NF κ B在决定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发挥重要作用。我们提出本申请的以下具体目标：1）。确定PARP在实验性胰腺炎和体外CCK刺激的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中的作用。 3)在实验性胰腺炎和体外CCK刺激的胰腺腺泡中，确定半胱天冬酶在坏死、凋亡和胰蛋白酶活化中的作用。 4）。确定NF κ B在实验性胰腺炎和体外用CCK刺激的胰腺腺泡中坏死和凋亡中的作用。 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、半胱天冬酶和胰蛋白酶的胰腺内活化、细胞色素c释放、线粒体膜电位、ATP水平和NF κ B活化，通过使用蛋白质印迹和凝胶位移分析、酶和荧光测定。 在拟议的具体目标的实验结果将划定的关键分子机制，调节坏死和凋亡的急性胰腺炎，这将导致新的治疗策略，以治疗疾病。

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.