Molecular Mechanisms of Acute Pancreatitis

描述(申请人提供)：胰腺炎是一个重要的临床问题，治疗仍在很大程度上支持。我们已经确定转录因子NF-kappaB是一种在疾病早期被激活的分子，有助于启动炎症级联反应。 具体目的1是确定核因子-kappaB激活在急性胰腺炎中的机制和作用。我们将通过腺病毒表达活性和显性负性亚基在胰腺内直接激活和抑制核因子-kappaB，从而扩大我们对核因子-kappaB激活的意义的理解。我们假设，核因子-kappaB发挥促炎作用，并特异性地驱动两种重要的趋化因子mob-1和gro-α的表达。我们将通过腺病毒介导的活性亚基的传递和寡核苷酸导向的微阵列分析相结合的方法，在胰腺腺泡细胞中鉴定由核因子-kappaB激活的其他基因。我们还将使用腺病毒方法直接激活腺泡细胞内的胰蛋白酶，来检验胰蛋白酶激活能够激活腺泡细胞内的核因子-kappaB的假设。 具体目标2是确定在急性胰腺炎病程早期诱导的基因。为了进一步确定在急性胰腺炎中重要的新分子，我们将利用寡核苷酸导向的微阵列来分析急性胰腺炎快速发作模型中的基因表达。我们将特别专注于转录因子和炎症介质的基因搜索，因为这些可能成为疾病治疗的目标。 具体目的#3是确定转录因子EGR-1在急性胰腺炎中的机制和作用。初步实验表明，在雨蛙素诱导的急性胰腺炎的早期，转录因子EGR-1被高度诱导。EGR-1在其他炎症性疾病中发挥重要作用，是已知的组织因子(TF)调节因子。我们将确定该转录因子是否在其他急性胰腺炎模型中也被诱导，研究其调控的细胞机制，并确定其激活与急性胰腺炎严重程度之间的关系。我们还将测试EGR-1驱动胰腺中TF表达的假设，并确定这一重要转录因子的其他靶点。 具体目的#4是确定促炎效应因子、MOB-1、GRO-α、TF和CGRP在急性胰腺炎中的作用。在用高浓度雨蛙素治疗动物后被鉴定为上调的促炎分子中，根据它们在其他疾病中的已知功能，我们选择了这四种分子，它们特别可能导致急性胰腺炎的严重程度。具体地说，我们将检验这样的假设，即MOB-1和GRO-α分别对T细胞和中性粒细胞的化学吸引起重要作用，TF对启动胰腺内的血液凝固很重要，腺泡细胞中CGRP的表达有助于胰腺和全身血流的改变。

DESCRIPTION (provided by applicant): Pancreatitis is an important clinical problem for which treatment is still largely supportive. We have identified the transcription factor NF-KappaB as a molecule that is activated early in the disease contributing to the initiation of an inflammatory cascade. Specific aim #1 is to determine the mechanisms and role of NF-KappaB activation in acute pancreatitis. We will extend our understanding of the significance of NF-KappaB activation by directly activating and inhibiting NF-KappaB within the pancreas using adenoviral expression of active and dominant negative subunits. We hypothesize that NF-KappaB plays a pro-inflammatory role and specifically drives expression of mob-1 and gro-alpha, two important chemokines. We will identify other genes activated by NF-KappaB within pancreatic acinar cells using a combination of adenovirus mediated delivery of an active subunit and oligonucleotide-directed microarray analysis. We will also test the hypothesis that trypsin activation is able to activate NF-KappaB within acinar cells using an adenoviral approach to directly active trypsin within acinar cells. Specific aim #2 is to identify the genes that are induced early in the course of acute pancreatitis. To further identify new molecules that are important in acute pancreatitis, we will utilize oligonucleotide directed microarrays to profile gene expression in rapid onset models of acute pancreatitis. We will specifically focus our search on genes for transcription factors and inflammatory mediators, as these may become targets of therapy for the disease. Specific aim #3 is to determine the mechanisms and role of the transcription factor EGR-1 in acute pancreatitis. Preliminary experiments have shown that the transcription factor EGR-1 is highly induced early in the course of caerulein induced acute pancreatitis. EGR-1 plays an important role in other inflammatory diseases and is a know regulator of tissue factor (TF). We will determine if this transcription factor is also induced in other models of acute pancreatitis, investigate the cellular mechanisms involved in its regulation, and determine the relationship between its activation and the severity of acute pancreatitis. We will also test the hypothesis that EGR-1 drives expression of TF in the pancreas, and identify other targets of this important transcription factor. Specific aim #4 is to determine the roles of pro-inflammatory effectors, mob-1, gro-alpha, TF and CGRP in acute pancreatitis. Of the pro-inflammatory molecules identified as up-regulated after treatment of animals with high concentrations of caerulein, we have selected these four as being especially likely to contribute to the severity of acute pancreatitis based upon their known functions in other diseases. Specifically, we will test the hypothesis that mob-1 and gro-alpha are important for chemoattraction of T-cells and neutrophils respectively, that TF is important for initiating blood coagulation in the pancreas, and that CGRP expression in acinar cells contributes to alterations in pancreatic and systemic blood flow.