THE INFLAMMATORY RESPONSE OF THE PANCREATIC ACINAR CELL

描述（由申请人提供）：胰腺腺泡细胞作为 用于确定蛋白质合成机制的细胞生物学模型， 由神经体液因子调节的运输和分泌。相反， 已知胰腺腺泡细胞在病理条件下， 胰腺炎和胰腺癌。我们有初步数据显示， 胰腺腺泡细胞的关键激动剂，激素胆囊收缩素（CCK）， 引起细胞内信号（即转录因子和 激酶）调节促炎细胞因子的产生， 趋化因子我们也有初步数据显示胰腺腺泡 细胞对细胞因子如肿瘤坏死因子（TNF-α）应答， 这种药物改变了CCK对细胞内信号的作用， 胰腺腺泡细胞介导炎症的产生， 分子（细胞因子、粘附分子等）。对于本申请， 我们假设在胰腺腺泡细胞中CCK激活细胞内 由TNF-α调节的信号介导炎性细胞因子的表达， 分子和胰腺炎。本申请的具体目的 （1）.确定CCK和TNF-α单独和联合使用的作用 对转录因子NF-κ-B和 AP-1，体外胰腺腺泡细胞。（二）、确定细胞内 参与介导CCK和TNF-α影响的信号系统 转录因子激活（三）、确定TNF-α对 胆囊收缩素诱导胰腺腺泡细胞表达细胞因子。（四）、确定 特定NF-κ-B蛋白（即p65、p50和c-Rel）在 调节胰腺腺泡细胞中细胞因子的表达。（五）、 确定特异性NF-κ-B蛋白在调节 胰腺细胞因子表达和炎症及细胞死亡反应 实验性胰腺炎本申请中的体外实验将使用 用胶原酶消化大鼠或小鼠胰腺制备腺泡细胞。 体内实验将使用各种NF-κ-B基因缺失的小鼠 蛋白质以及野生型对照。将使用RT-PCR测定细胞因子， 北方和Western印迹分析和免疫细胞化学。细胞内 测量的信号将包括转录因子激活、激酶 活性和第二信使用凝胶迁移分析、蛋白质印迹法 分析、酶测定、放射免疫测定和细胞内Ca 2 + 浓度的胰腺炎的指标将包括形态学 （包括免疫组织化学）技术和生化测定 转录因子激活、细胞因子表达和胰蛋白酶激活， 胰腺组织本发明中描述的实验的结果如下： 应用程序将使我们能够提出CCK如何与细胞因子一起 调节其作用，作用于胰腺腺泡细胞， 实验性胰腺炎

DESCRIPTION (provided by applicant): The pancreatic acinar cell has served as a cell biologic model for determining the mechanisms of protein synthesis, transport and secretion regulated by neurohumoral agents. In contrast, little is known about the pancreatic acinar cell in pathologic conditions such as pancreatitis and pancreatic cancer. We have preliminary data showing that the key agonist for pancreatic acinar cells, the hormone cholecystokinin (CCK), causes activation of intracellular signals (i.e. transcription factors and kinases) that regulate the production of proinflammatory cytokines and chemokines. We also have preliminary data showing that the pancreatic acinar cell responds to cytokines such as tumor necrosis factor (TNF-alpha) and that this agent modifies the effect of CCK on the intracellular signals in the pancreatic acinar cell that mediate the generation of the inflammatory molecules (cytokines, adhesion molecules, etc.). For the present application, we hypothesize that in the pancreatic acinar cell CCK activates intracellular signals modulated by TNF-alpha that mediate the expression of the inflammatory molecules and pancreatitis. The specific objectives for the present application are: (1). Determine the effects of CCK and TNF-alpha alone and in combination on activation and composition of the transcription factors, NF-kappa-B and AP-1, in pancreatic acinar cells in vitro. (2). Determine the intracellular signaling systems involved in mediating the effects of CCK and TNF-alpha on transcription factor activation. (3). Determine the effect of TNF-alpha on CCK-induced expression of cytokines in pancreatic acinar cells. (4). Determine the roles of specific NF-kappa-B proteins (i.e. p65, p50 and c-Rel) in regulating cytokine expression in pancreatic acinar cells in vitro. (5). Determine the roles of the specific NF-kappa-B proteins in regulating pancreatic cytokine expression and the inflammatory and cell death responses of experimental pancreatitis. In vitro experiments in this application will use acinar cells prepared by collagenase digestion of pancreas from rat or mouse. In vivo experiments will use mice with genetic deletions of various NF-kappa-B proteins as well as wild-type controls. Cytokines will be assayed using RT-PCR, Northern and Western blot analyses, and immunocytochemistry. Intracellular signals measured will include transcription factor activation, kinase activities, and second messengers measured with gel shift assay, Western blot analysis, enzyme assays, radioimmunoassay, and intracellular Ca2+ concentrations. Measures of pancreatitis will include both morphologic (including immunohistochemical) techniques and biochemical determinations of transcription factor activation, cytokine expression, and trypsin activation in the pancreatic tissue. The results of the experiments described in the present application will allow us to propose how CCK, together with a cytokine modulating its effects, acts on the pancreatic acinar cell to cause experimental pancreatitis.