Mechanisms underlying pancreatitis

胰腺炎的潜在机制

• 批准号: 8293385
• 负责人: Gregory P Botta
• 金额: $ 4.2万
• 依托单位: DREXEL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8293385
• 关键词: 3-Dimensional; Acinus organ component; Acute; Adhesions; Adult; Affect; Alcoholic Pancreatitis; Alcohols; Antibodies; Apoptosis; Apoptotic; Biological Models; Cell Adhesion Molecules; Cells; Cessation of life; Characteristics; Clinical; Confocal Microscopy; Culture Media; DNA Damage; DNA Microarray Chip; Desmoplastic; Development; Disintegrins; Duct (organ) structure; Ductal; Ductal Epithelial Cell; Ductal Epithelium; Elasticity; Environment; Epithelial; Epithelial Cells; Ethanol; Event; Exhibits; Exposure to; Extracellular Matrix; Future; Generations; Growth Factor; Heavy Drinking; Hemorrhagic Shock; Human; Hydrogels; IL8 gene; In Vitro; Induction of Apoptosis; Inflammation Mediators; Inflammatory; Injury; Integrins; Lipid Peroxidation; Mammary gland; Measures; Microarray Analysis; Modeling; Morphogenesis; Pain; Pancreas; Pancreatic duct; Pancreatitis; Pathogenesis; Pathology; Pathway interactions; Physiological; Production; Reaction; Reactive Oxygen Species; Respiratory distress; Rodent; Role; Signal Transduction; Simulate; Staining method; Stains; Testing; Time; Validation; alcohol exposure; alcohol response; annexin A5; caspase-3; chemokine; in vitro Model; in vivo; matrigel; nestin protein; neutralizing antibody; novel; polyacrylamide gels; research study; response; scaffold; stellate cell; viscoelasticity

DESCRIPTION (provided by applicant): The most common cause of Pancreatitis is excessive alcohol intake which has the potential to induce reactive oxygen species (ROS) leading to DNA damage and apoptosis. In addition, characteristic pathological responses of pancreatitis, including inflammatory chemokines and a stiff, desmoplastic microenvironment may also contribute to this increased oxidative potential in pancreatic ductal cells. ROS can cause a severe inflammatory reaction instigating severe pain, hemorrhagic shock, acute respiratory distress, or death, however, no in vitro human model yet exists to understand this pathology. As such, we have begun to develop a novel, three-dimensional, human pancreatic duct model composed of nestin-positive, immortalized, pancreatic epithelial cells, Matrigel scaffolds, predetermined culture media, and specific growth factors. This model is characterized via confocal microscopy for cellular organization, polarization, and functionality similar to human mammary epithelial acini. Secondly, much progress has been made in understanding pancreatic stellate cell reactions to ethanol, but less is understood of the pancreatic epithelial cell response. Thus, we will analyze the effect of increasing concentrations of ethanol on ROS induction within these human pancreatic ducts via fluorescent staining and lipid peroxidation as well as the subsequent DNA damage response and apoptotic initiation events via the double strand break markers (H2AX and (SP1101 and Caspase-3, Bim, and Annexin V, respectively. Thirdly, pancreatic stellate cells have also been implicated in ROS production via inflammatory mediators, however, little has been advanced in the pancreatic epithelial cell response. As such, we will examine the direct initiation of ROS within the human pancreatic duct model via an inflammatory chemokine regularly found within the pancreatitis environment, IL-8. Through treatment with IL-8, we will determine the human pancreatic epithelial cell duct ROS-induced DNA damage and apoptosis response. Lastly, we will continue to verify the extent of ROS production within human pancreatic epithelial ducts by a stiffening extracellular matrix. By blocking pancreatic-specific integrin (3, (4 and (6 through antibodies/disintegrins and by DNA microarray validation of their expression, we will understand how exogenous elastic stiffness can affect ROS potential internal to pancreatic epithelial cells. We anticipate that the development of our in vitro, 3-D, human pancreatic epithelial duct model along with each of the three independent experiments will uncover parts of the etiologic pathway leading to pancreatitis in a human-relevant model and offer possible targets for future clinical therapy.

描述（由申请人提供）：胰腺炎最常见的原因是过量饮酒，这有可能诱发活性氧（ROS），导致 DNA 损伤和细胞凋亡。此外，胰腺炎的特征性病理反应，包括炎症趋化因子和僵硬的促纤维增生微环境，也可能导致胰腺导管细胞氧化电位增加。 ROS 可引起严重的炎症反应，引发剧烈疼痛、失血性休克、急性呼吸窘迫或死亡，然而，尚无体外人体模型来了解这种病理学。因此，我们已经开始开发一种新型的三维人类胰管模型，由巢蛋白阳性、永生化的胰腺上皮细胞、基质胶支架、预定的培养基和特定的生长因子组成。该模型通过共聚焦显微镜进行细胞组织、偏振和功能的表征，类似于人类乳腺上皮腺泡。其次，在了解胰腺星状细胞对乙醇的反应方面已经取得了很大进展，但对胰腺上皮细胞反应的了解却很少。因此，我们将通过荧光染色和脂质过氧化分析增加乙醇浓度对这些人胰管内 ROS 诱导的影响，以及通过双链断裂标记（H2AX 和（SP1101 和分别是 Caspase-3、Bim 和膜联蛋白 V 第三，胰腺星状细胞也通过炎症介质参与 ROS 的产生，然而，在胰腺上皮细胞中进展甚微。因此，我们将通过胰腺炎环境中常见的炎症趋化因子 IL-8 来检查人胰腺导管模型中 ROS 的直接启动，通过使用 IL-8 进行治疗，我们将确定人胰腺上皮细胞导管。 ROS 诱导的 DNA 损伤和细胞凋亡反应最后，我们将继续通过硬化细胞外基质验证人胰腺上皮导管内 ROS 产生的程度。通过抗体/解整合素和 DNA 微阵列验证其表达来阻断胰腺特异性整合素 (3, (4 和 (6))，我们将了解外源弹性刚度如何影响胰腺上皮细胞内部的 ROS 潜力。我们预计我们的体外 3D 人类胰腺上皮导管模型以及三个独立实验中的每一个都将在人类相关模型中揭示导致胰腺炎的部分病因途径，并为未来的临床治疗提供可能的目标。