The Role of Trypsin in Pancreatitis

描述(由申请人提供)：本提案的中心目标是通过腺泡内细胞内胰蛋白酶原直接激活来建立和表征急性和慢性胰腺炎的新模型。最近在遗传性胰腺炎患者中发现的胰蛋白酶原和胰酶抑制物突变支持了这样的假设，即胰腺酶原不适当地激活胰腺内的活性酶启动了炎症过程。因此，胰酶的过早激活被认为是胰腺炎发生的第一步。然而，目前的急性胰腺炎动物模型涉及到许多具有非特异性作用的治疗方法，除了激活胰蛋白酶外，还会触发许多信号通路。慢性胰腺炎的情况甚至不太清楚，因为这种疾病还没有可靠的动物模型。具体目的1是通过胰腺腺泡细胞内胰酶的直接激活来诱导大鼠急性胰腺炎。我们将首先使用腺病毒介导的突变胰蛋白酶原(AdPACE-Try)的基因转移到大鼠胰腺，我们开发的这种突变的胰酶原已经被证明在腺泡细胞中被激活。我们假设，体内将这种病毒运送到胰腺会激活胰酶，从而引发胰腺炎。具体目的#2是确定细胞内胰酶是否诱导核因子-kB的激活，以及核因子-kB在胰酶诱导的急性胰腺炎中的作用。我们之前已经证明，核因子-KB的激活不会导致胰蛋白酶原的激活。在这里，我们将测试腺泡细胞内的胰蛋白酶活性是否可以使用AdPACE激活核因子-KB-尝试在细胞内激活胰蛋白酶，并探讨相关的机制。我们将通过联合应用表达kB-cc的腺病毒来进一步研究胰酶激活核因子-kB-kB在炎症级联反应中的重要性，这将阻断核因子-kB激活。具体目的#3是通过直接调节腺泡内胰酶活性建立慢性胰腺炎模型。我们将通过直接调节腺泡内胰酶活性来建立慢性胰腺炎模型。为此，弹性酶启动子驱动的三苯氧胺可调节的Cre-er转基因小鼠将与loxP-GFP侧翼Pace-try转基因小鼠杂交。他莫昔芬激活Cre后，将移除一个允许表达活性胰酶的停止序列。我们预计大剂量的他莫昔芬将诱导高胰酶活性，从而导致重症急性胰腺炎，而小剂量的他莫昔芬将导致部分胰腺损伤和长期的胰腺损伤，从而导致慢性胰腺炎。

DESCRIPTION (provided by applicant): The central goal of this proposal is to establish and characterize novel models of acute and chronic pancreatitis by direct intra-acinar cell trypsinogen activation. Recent discoveries of trypsinogen and trypsin inhibitor mutations in patients with hereditary pancreatitis support the hypothesis that an inappropriate activation of pancreatic zymogens to active enzymes within the pancreas starts the inflammatory process. Thus premature activation of trypsin is believed to be the initial step in the development of pancreatitis. However, current animal models of acute pancreatitis involve treatments that have many non-specific effects and trigger numerous signaling pathways in addition to activating trypsin. The situation for chronic pancreatitis is even less clear, as there are no reliable animal models of this disease. Specific aim #1 is to induce acute pancreatitis by direct trypsin activation within pancreatic acinar cells in rats. We will initially use adenoviral mediated gene transfer into the rat pancreas of a mutant trypsinogen (AdPACE-TRY) which we developed and have shown to become activated within the acinar cells. We hypothesize that delivery of this virus to the pancreas in vivo will active trypsin and thereby initiate pancreatitis. Specific aim #2 is to determine whether intracellular trypsin induces NF-KB activation and the role of NF-KB in trypsin induced acute pancreatitis. We have previously shown that NF-KB activation does not cause trypsinogen activation. Here we will test if trypsin activity within the acinar cell can activate NF-KB using AdPACE-TRY to activate trypsin intracellularly and investigate the mechanisms involved. We will further examine the importance of trypsin activation of NF-KB in the initiation of the inflammatory cascade by co-administering an adenovirus expressing kB-cc which will block NF-KB activation. Specific aim #3 is to establish a chronic pancreatitis model by directly regulating intra-acinar trypsin activity. We will create a chronic pancreatitis model by directly regulating intra-acinar trypsin activity. For this purpose, an elastase promoter driven tamoxifen regulatable Cre-Er transgenic mouse will be crossed with a loxP- GFP flanked PACE-TRY transgenic mouse. Tamoxifen activation of Cre will then remove a stop sequence allowing the expression of the active trypsin. We expect that a high dose tamoxifen will induce high trypsin activity and thus cause severe acute pancreatitis and that a low dose tamoxifen will induce partially pancreatic injury and prolonged pancreatic damage that will result in chronic pancreatitis.