Cell Death and Autophagy in Chronic Pancreatitis

慢性胰腺炎中的细胞死亡和自噬

• 批准号: 10266019
• 负责人: ANNA S. GUKOVSKAYA
• 金额: --
• 依托单位: VA GREATER LOS ANGELES HEALTHCARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10266019
• 关键词: Acinar Cell; Age; Alleles; Atrophic; Autophagocytosis; Birth; Caerulein; Cell Death; Cell Survival; Cells; Cessation of life; Complication; Cytoplasmic Protein; Data; Development; Disease; Excess Mortality; Exocrine pancreas; Exocrine pancreatic insufficiency; Experimental Models; Functional disorder; Genetic; Genetic Models; Gland; Goals; Health Care Costs; Histologic; Human; Impairment; Investigation; Link; Long-Term Effects; Lysosomes; Malignant neoplasm of pancreas; Mediating; Mediator of activation protein; Membrane Proteins; Modality; Modeling; Modification; Morbidity - disease rate; Multiprotein Complexes; Mus; Mutation; Necrosis; Organelles; Orthologous Gene; Pancreas; Pancreatitis; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Pharmacology; Phosphotransferases; Play; Predisposing Factor; Process; Protein Inhibition; Proteins; Quality of life; RIPK3 gene; Risk; Risk Factors; Role; SPINK1 gene; Stress; Testing; Therapeutic; Tissues; Transgenic Mice; Up-Regulation; Vacuole; Veterans; acute pancreatitis; cell type; chronic pancreatitis; clinically relevant; effective therapy; genetic approach; human disease; inhibitor/antagonist; lysosomal proteins; mouse model; novel; patient population; prevent; promoter; protein aggregation; protein function; restoration; transcription factor

Chronic pancreatitis (CP) is a disease of the exocrine pancreas common among Veterans patient population, the pathogenesis of which remains obscure and for which there are no specific or effective treatments. The disease is burdened with poor quality of life of the patients and excess mortality, considerable health care costs, and is a major risk factor for the deadly pancreatic cancer. Among a few known genetic factors predisposing to pancreatitis are mutations in a protein called SPINK1, which increase the risk of chronic pancreatitis 20- to 40-fold. The function of this protein is not clear; mice with genetic deletion of Spink3, the mouse ortholog of SPINK1, die shortly after birth, thus preventing investigation of long-term effects of SPINK deficiency. To overcome this obstacle, we developed transgenic mice in which one of the two Spink3 alleles ablated in Spink3-/- mice is replaced by human SPINK1. In these mice (termed SPINK1- in) the expression of SPINK is partially restored under control of its endogenous promoter. We found that such restoration rescues Spink3-/- mice from lethality; and further, that SPINK1-in mice develop spontaneous pancreatitis, thus representing a new genetic model of CP. We propose to use this new genetic model, as well as the commonly used repetitive-cerulein experimental model to elucidate the pathogenic mechanism of CP, in particular, the death pathways of acinar cells, the main cell type of the exocrine pancreas. Acinar cell death is a major complication of CP, leading to parenchymal loss, pancreas atrophy, and exocrine insufficiency. Maintaining acinar cell survival to prevent or reduce parenchymal loss in CP is of paramount importance as a therapeutic approach. However, essentially nothing is known on the modalities and mechanisms of acinar cell death in CP. Our preliminary results indicate that a distinct type of programmed necrosis, termed necroptosis, is the major modality of acinar cell death in CP. The data further indicate that upregulation of kinase RIP3 underlies necroptosis in CP models. Autophagy is the principal cellular degradative, lysosome-driven mechanism; it mediates cell adaptation to stresses by removing damaged organelles and protein aggregates toxic for the cell. SPINK1-in mice very early develop massive accumulation of autophagic vacuoles in acinar cells, an indication of impaired autophagic flux. Our preliminary results further suggest that underlying mechanisms involve lysosomal dysfunction caused by loss of LAMP2, a major lysosomal membrane protein. Autophagy is a key cell survival mechanism; in particular, it eliminates a multiprotein complex termed necrosome, which mediates necroptosis. However, whether autophagy is impaired in CP, and whether it plays a role in acinar cell death in CP, has not been studied. We propose to use the genetic and experimental models of CP to elucidate the mechanisms of autophagic/lysosomal dysfunction and acinar cell death, and the link between these pathologies in CP. The central hypothesis of this proposal is that the major pathway of acinar cell death in CP is necroptosis, mediated by autophagic/lysosomal dysfunction. Pharmacologic and genetic approaches to inhibit necroptosis and to restore efficient autophagy will prevent acinar cell death and ameliorate CP. We propose the following Specific Aims: 1) Determine the mechanisms of acinar cell death in the genetic and experimental models of CP; 2) Determine the mechanisms of autophagic/lysosomal dysfunction in CP models; 3) Determine the role of impaired autophagy in acinar cell death in CP models.

慢性胰腺炎（CP）是退伍军人患者人群中常见的胰腺外分泌疾病， 其发病机理仍然不清楚，并且没有特异性或有效的治疗方法。的 疾病负担着患者的生活质量差和过高的死亡率，相当大的医疗保健费用， 是致命胰腺癌的主要危险因素。 在一些已知的诱发胰腺炎的遗传因素中，有一种叫做SPINK1的蛋白质的突变， 这会使慢性胰腺炎的风险增加20到40倍。这种蛋白质的功能尚不清楚; SPINK3基因缺失，SPINK1的小鼠直系同源物，出生后不久死亡，因此阻止了对SPINK3的研究。 SPINK缺乏症的长期影响。为了克服这一障碍，我们开发了转基因小鼠，其中一个 在Spink3-/-小鼠中消除的两个Spink3等位基因中的一个被人SPINK1取代。在这些小鼠（称为SPINK1- 在）中，SPINK的表达在其内源启动子的控制下部分恢复。我们发现， 恢复挽救了Spink3-/-小鼠的致死性;此外，SPINK 1-小鼠自发地 胰腺炎，因此代表了一种新的CP遗传模型。 我们建议使用这种新的遗传模型，以及常用的重复-雨蛙素实验 模型，以阐明CP的致病机制，特别是腺泡细胞的死亡途径，主要的 胰腺外分泌的细胞类型。腺泡细胞死亡是CP的主要并发症，导致实质损失， 胰腺萎缩和外分泌不足。维持腺泡细胞存活以防止或减少实质性 CP的丧失作为治疗方法是至关重要的。然而，基本上没有什么是已知的， CP中腺泡细胞死亡的方式和机制。我们的初步结果表明，一种独特的 程序性坏死，称为坏死性凋亡，是CP中腺泡细胞死亡的主要形式。所述数据进一步 表明激酶RIP3上调是CP模型中坏死性凋亡的基础。 自噬是主要的细胞降解，溶酶体驱动的机制;它介导细胞适应 通过去除受损的细胞器和对细胞有毒的蛋白质聚集体来应激。SPINK1-小鼠极早期 在腺泡细胞中出现大量自噬空泡积聚，表明自噬通量受损。 我们的初步结果进一步表明，潜在的机制涉及溶酶体功能障碍， LAMP2（一种主要的溶酶体膜蛋白）的丢失。自噬是一种关键的细胞存活机制;特别是， 它消除了称为坏死体的多蛋白复合物，该复合物介导坏死性凋亡。但无论 自噬在CP中受损，以及它是否在CP的腺泡细胞死亡中起作用还没有研究。我们 建议使用CP的遗传和实验模型来阐明自噬/溶酶体的机制 功能障碍和腺泡细胞死亡，以及CP中这些病理之间的联系。 该建议的中心假设是CP中腺泡细胞死亡的主要途径是坏死性凋亡， 由自噬/溶酶体功能障碍介导。抑制坏死性凋亡的药理学和遗传学方法 恢复有效的自噬将防止腺泡细胞死亡，改善CP。 我们提出以下具体目的：1）确定腺泡细胞死亡的机制，在遗传 CP的自噬/溶酶体功能障碍的机制 3）确定受损的自噬在CP模型中腺泡细胞死亡中的作用。