MECHANISMS OF LIVER REPAIR AFTER BILIARY RECONSTRUCTION

胆道重建后肝脏修复的机制

• 批准号: 2146088
• 负责人: THOMAS F TRACY
• 金额: $ 9.55万
• 依托单位: SAINT LOUIS UNIVERSITY
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2146088
• 关键词: DNA replication; Kupffer's cell; RNase protection assay; autocrine; cell cycle proteins; cell growth regulation; cholestasis; chronic disease /disorder; gene expression; growth factor receptors; hepatocyte growth factor; immunocytochemistry; in situ hybridization; injury; laboratory rat; liver cells; liver regeneration; mitogens; morphology; protein biosynthesis; protooncogene; receptor expression; transforming growth factors; tumor necrosis factor alpha; western blottings

The broad goal of this research is to establish the physiologic significance of mitogenic growth factors in liver repair after chronic injury. This proposal focuses on the potential roles in repair for transforming growth factors a&B (TGFalpha &beta), hepatocyte growth factor (HGF), and Kupffer cell derived tumor necrosis factor alpha (TNFalpha) which are known to regulate liver regeneration after acute injury. There are five specific aims. Aim 1: Determine TGFalpha&Beta, HGF, and TNFalpha gene expression in the liver during chronic, cholestatic injury and repair. The simultaneous expression of protooncogene mRNAs coding for receptors for TGFalpha and HGF, c-erbB and c-met, will be determined and protein levels will be measured. Cholestasis will be initiated in rats by vessel loop suspension of the common bile duct for increasing periods of injury; release of the loop allows biliary decompression to simulate reconstruction and initiate repair. mRNA levels will be quantified by ribonuclease protection assays and protein by Western blots. Aim 2: Localize the cellular sources of HGF and TGF alpha & Beta mRNA and proteins during repair after chronic injury b a combination of in situ hybridization and immunohistochemical techniques. Aim 3: Related changes in steady state levels of growth factor and receptor expression to in vivo hepatocellular DNA synthesis by immunohistochemistry for proliferating cell nuclear antigen. Important modulator os the regenerative response will be studied during repair after Kupffer cell blockade by the injection of gadolinium and after endotoxin restriction by the administration of Polymyxin B. Aim 4: Examine functional and proliferative changes to in vitro TGFalpha and HGF stimulation in hepatocytes isolated after increasing periods of cholestatic injury. Hepatocytes will be extracted and purified by centrifugal elutriation and in vitro morphology, DNA synthesis, and albumin synthesis will be measured. Aim 5: Establish the autocrine mechanism(s) of growth factor stimulation by TGFalpha on normal and injured hepatocytes by measuring DNA synthesis, the hepatocytes expression of c-erbB and, the levels of TGFalpha mRNA. These studies are designed to test the hypothesis that after acute injury, liver repair and regeneration share similarities in the growth factor control of hepatocyte proliferation. However, because regeneration yields a normal liver, and repair often lead to scar, quantitative differences in either growth factors and their receptors, or the hepatocyte response to growth factor stimulation must exist to slow the course, and inhibit the quality of repair. Data from this combined in vivo and in vitro approach should provide insights into the role and mechanism of action of growth factors in chronic liver injury and repair.

这项研究的广泛目标是建立生理 慢性后，有丝分裂生长因子在肝修复中的重要性 受伤。 该提议着重于维​​修的潜在作用 改变生长因子A＆B（TGFALPHA和BETA），肝细胞生长 因子（HGF）和Kupffer细胞衍生的肿瘤坏死因子α （tnfalpha）已知会在急性后调节肝脏再生 受伤。 有五个具体目标。 目标1：确定TGFALPHA和BETA，HGF和TNFALPHA基因表达 慢性，胆汁淤积损伤和修复期间的肝脏。 同时 protooncogene mRNA的表达，编码用于TGFALPHA的受体和 HGF，C-ERBB和C-MET将确定，蛋白质水平将为 测量。 胆汁淤积将通过容器环在大鼠中启动 暂停普通胆管的伤害周期； 循环的释放允许胆道减压模拟 重建和启动维修。 mRNA水平将通过 核糖核酸酶保护测定法和蛋白质通过蛋白质印迹。 AIM 2：定位HGF和TGF alpha＆beta mRNA的细胞来源以及 慢性损伤后修复过程中的蛋白质B组合原位 杂交和免疫组织化学技术。 目标3：生长因子的稳态水平的相关变化和 受体表达对体内肝细胞DNA合成的表达 免疫组织化学用于增殖细胞核抗原。 重要的 调节器OS将在维修期间研究再生响应 通过注射gadolinium和之后，Kupffer细胞阻塞后 通过施用多粘蛋白B的内毒素限制。 AIM 4：检查体外TGFALPHA的功能和增殖变化 在增加周期后分离的肝细胞中的HGF刺激 胆汁淤积损伤。 肝细胞将被提取和纯化 离心放松和体外形态，DNA合成和 将测量白蛋白合成。 目标5：建立生长因子刺激的自分泌机制 通过TGFALPHA在正常和受伤的肝细胞上测量DNA合成， C-ERBB的肝细胞表达和TGFALPHA mRNA的水平。 这些研究旨在检验以下假设 受伤，肝修复和再生在增长方面具有相似之处 肝细胞增殖的因子控制。 但是，因为 再生会产生正常的肝脏，并且修复通常会导致疤痕， 两种生长因子及其受体的定量差异， 或必须存在肝细胞对生长因子刺激的反应 减慢路线，并抑制维修质量。 来自此的数据 结合体内和体外方法应提供有关 生长因子在慢性肝损伤中的作用和机制 和维修。