Cellular Mechanisms of Matrix Regulation in Liver Repair

肝脏修复中基质调节的细胞机制

• 批准号: 6543041
• 负责人: THOMAS F TRACY
• 金额: $ 32.41万
• 依托单位: RHODE ISLAND HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-05-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6543041
• 关键词: Kupffer's cell; RNase protection assay; SDS polyacrylamide gel electrophoresis; cell growth regulation; collagenase; cytokine; enzyme activity; extracellular matrix; fibrosis; gene expression; growth factor receptors; hepatocyte growth factor; immunocytochemistry; injury; laboratory rat; laser capture microdissection; liver regeneration; macrophage; messenger RNA; metalloendopeptidases; northern blottings; polymerase chain reaction; postmortem; tissue /cell culture; tissue inhibitor of metalloproteinases; Kupffer's cell; RNase protection assay; SDS polyacrylamide gel electrophoresis; cell growth regulation; collagenase; cytokine; enzyme activity; extracellular matrix; fibrosis; gene expression; growth factor receptors; hepatocyte growth factor; immunocytochemistry; injury; laboratory rat; laser capture microdissection; liver regeneration; macrophage; messenger RNA; metalloendopeptidases; northern blottings; polymerase chain reaction; postmortem; tissue /cell culture; tissue inhibitor of metalloproteinases

DESCRIPTION (provided by applicant)At critical points in chronic liver injury the potential for liver repair is lost. Timely surgical and medical treatment however, initiates liver repair interrupting the cycle of inflammation and hepatic fibrosis. The subsequent cellular regulation of matrix protein degradation and resolution of injury dependent fibrosis is unknown. The long-term goals of our studies are to define the molecular mechanisms required for ordered matrix resorption and liver repair without scar. Specific aims have been designed to test the hypothesis that repair, resolution of scar, and the restoration of hepatic architecture depend on coordinated regulatory mechanisms of matrix degradation through matrix metalloproteinase (MMP) and tissue inhibitor of MMP (TIMP) gene expression, cellular localization and most importantly, biologic activity. Further, we propose that Kupffer cells (KC), the resident macrophage population, have a central role in the inflammatory and fibrogenic regulation of liver repair after chronic injury. Specific Aim 1 addresses the molecular and cellular mechanisms of reversible hepatic fibrosis and those of delayed matrix resorption. A unique rat model of reversible biliary obstruction will simulate early, intermediate, and late, near end stage cholestatic liver injury. This model allows biliary decompression to initiate liver repair. [The effect of injury duration on key matrix (collagen I and III, laminin), MMP (1,2,8,9) and TIMP (1,2) mRNA and protein expression will be measured in whole liver and in [individual] cells using new methods of laser capture microdissection. [These data will demonstrate the mechanism and physiology of matrix resorption through measurement of MMP activity by in-gel and in-situ zymography]. Specific Aim 2 tests the hypothesis that KC are in vivo regulators of matrix metabolism. Through strategies of KC depletion/ inactivation by gadolinium or Dexa-Man10-HSA targeted dexamethasone during repair, matrix resorption, regulatory cytokine profiles and MMP-TIMP activity will be localized in cells and measured. In Specific Aim 3 we will establish the effect of progressive injury on the in vitro capacity of isolated hepatic macrophages (MPh) to express specific MMP and fibrogenic/ antifibrogenic cytokine (TGFbeta, IL1, 6, 10) profiles during repair. KC inactivation and MMP stimulation/ suppression strategies will isolate the net collagenolytic activity of MPh. [In addition to the broad clinical implications for the treatment of cholestatic or other chronic liver diseases in infants, children, and adults, these studies may be the first to identify key molecular mechanisms for successful repair after progressive liver injury. They have the unique potential to yield targets for therapy to promote liver repair or rescue patients approaching end stage liver disease

描述（由申请人提供）在慢性肝损伤的关键点上丢失了肝脏修复的潜力。然而，及时的手术和医疗启动肝修复，中断炎症和肝纤维化的周期。随后的基质蛋白降解的细胞调节和依赖性纤维化的分辨率尚不清楚。我们研究的长期目标是定义有序基质吸收和肝修复所需的分子机制，而无需疤痕。特定目的旨在检验以下假设：修复，疤痕的分辨率以及肝结构的恢复取决于基质降解的协调调节机制通过基质金属蛋白酶（MMP）和MMP（TIMP）基因表达，细胞局部化以及最重要的生物学活性的MMP（TIMP）基因表达，MMP（TIMP）抑制剂的调节机制。此外，我们提出，居民巨噬细胞种群Kupffer细胞（KC）在慢性损伤后肝修复的炎症和纤维化调节中具有核心作用。具体目标1解决了可逆肝纤维化的分子和细胞机制以及延迟基质吸收的分子机制。可逆的胆道阻塞的独特大鼠模型将模拟早期，中间和晚期近端胆汁淤积性肝损伤。该模型允许胆道减压启动肝脏修复。 [损伤持续时间对密钥基质（胶原I和III，层粘连蛋白），MMP（1,2,8,9）和TIMP（1,2）mRNA和蛋白质表达的影响将在整个肝脏和[单个]细胞中使用激光捕获微分解的新方法进行测量。 [这些数据将通过凝胶和原位酶学测量MMP活性来证明基质吸收的机制和生理学]。具体目标2检验了KC是基质代谢的体内调节剂的假设。通过在修复过程中靶向地塞米松的KC消耗/灭活/灭活的策略，基质吸收，调节性细胞因子谱和MMP-TIMP活性将定位在细胞中并进行测量。在特定的目标3中，我们将建立进行性损伤对分离的肝巨噬细胞（MPH）体外能力的影响，以表达特定的MMP和纤维化/抗纤维细胞因子（TGFBETA，IL1、6、10）谱。 KC失活和MMP刺激/抑制策略将隔离MPH的净胶原式活性。 [除了对婴儿，儿童和成年人中胆固醇或其他慢性肝病的治疗具有广泛的临床意义外，这些研究可能是第一个识别进行性肝损伤后成功修复的关键分子机制的研究。它们具有产生治疗靶标的促进肝脏修复或营救患者接近末期肝病的患者的独特潜力