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.

这项研究的总体目标是建立生理学 有丝分裂生长因子在慢性肝损伤修复中的意义 受伤。 该提案重点关注修复中的潜在作用 转化生长因子 a&B (TGFalpha &beta)、肝细胞生长 因子 (HGF) 和库普弗细胞衍生的肿瘤坏死因子 α (TNFα) 已知可调节急性肝损伤后的肝再生 受伤。 有五个具体目标。 目标 1：确定 TGFalpha&Beta、HGF 和 TNFalpha 基因表达 肝脏在慢性、胆汁淤积性损伤和修复过程中。 同时进行的 编码 TGFα 受体的原癌基因 mRNA 的表达 将测定 HGF、c-erbB 和 c-met，并测定蛋白质水平 测量。 通过血管环在大鼠中引发胆汁淤积 悬吊胆总管以延长受伤时间； 释放循环允许模拟胆道减压 重建并开始修复。 mRNA 水平将通过以下方式量化 通过蛋白质印迹进行核糖核酸酶保护测定和蛋白质。 目标 2：定位 HGF 和 TGF α & Beta mRNA 的细胞来源并 慢性损伤后修复过程中的蛋白质b原位组合 杂交和免疫组织化学技术。 目标 3：生长因子和稳态水平的相关变化 受体表达对体内肝细胞DNA合成的影响 增殖细胞核抗原的免疫组织化学。 重要的 调节器操作系统的再生反应将在修复过程中进行研究 通过注射钆阻断库普弗细胞后 通过施用多粘菌素 B 限制内毒素。 目标 4：检查体外 TGFalpha 的功能和增殖变化 和 HGF 刺激在增加时间后分离的肝细胞 胆汁淤积性损伤。 肝细胞将被提取和纯化 离心淘洗和体外形态学、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 通过测量 DNA 合成，通过 TGFalpha 对正常和受损的肝细胞进行检测， 肝细胞c-erbB的表达和TGFα mRNA的水平。 这些研究旨在检验以下假设：急性发作后 损伤、肝脏修复和再生在生长方面有相似之处 肝细胞增殖的控制因素。 然而，因为 再生会产生正常的肝脏，而修复往往会导致疤痕， 生长因子及其受体的数量差异， 或者肝细胞对生长因子刺激的反应必须存在 减慢进程并抑制修复质量。 数据来自于此 体内和体外相结合的方法应该提供对 生长因子在慢性肝损伤中的作用及作用机制 和修复。