MANIPULATING HEPATOCYTE PROLIFERATION BY GENE TARGETING.

本RO 3提案的目的是检查细胞周期蛋白D1和其他细胞周期调节蛋白在培养和体内促进肝细胞增殖的能力。成熟的肝细胞保留了干细胞的功能，并可以在肝损伤后重新填充肝脏。先前的研究表明，细胞周期蛋白D1的上调在调控肝细胞通过细胞周期G1期的进展中起重要作用。为了检测细胞周期蛋白D1是否能够促进非丝裂原依赖性肝细胞增殖，我们构建了一种编码这种蛋白的复制缺陷型腺病毒（ADV-D1）。我们的初步研究表明，ADV-D1，而不是对照腺病毒，促进细胞周期的进展，在原代肝细胞生长因子的情况下，并诱导肝细胞增殖细胞外基质，通常是生长抑制。初步体内研究表明，ADV-D1有效地抑制了完整动物的肝细胞。这些结果表明，用ADV-D1转染肝细胞可以促进肝细胞增殖，而不依赖于细胞外信号，并保证作为促进肝再生的试剂进行测试。本研究的三个具体目的将研究细胞周期蛋白D1促进肝细胞生长的机制，并将研究其他G1期细胞周期蛋白和E2 F转录因子促进肝细胞周期进程的能力。这些研究旨在为肝细胞生长的调控提供独特的机制见解，并确定用于基因靶向策略的候选蛋白质，以治疗性地增强肝再生。

The aim of this RO3 proposal is to examine the ability of cyclin D1 and other cell cycle regulatory proteins to promote hepatocyte proliferation in culture and in vivo. Mature hepatocytes retain the ability to function as stem cells and can repopulate the live after hepatic injury. Previous studies have suggested that up-regulation of cyclin D1 plays an important role in governing progression of hepatocytes through G1 phase of the cell cycle. To test whether cyclin D1 is capable of promoting mitogen-independent hepatocyte proliferation, we have constructed a replication-defective adenovirus encoding this protein (ADV-D1). Our preliminary studies indicate that ADV-D1 but not a control adenovirus, promotes cell cycle progression in primary hepatocytes in the absence of growth factor, and induces hepatocyte proliferation on extracellular matrices that are normally growth-inhibitory. Pilot in vivo studies indicate that ADV-D1 effectively transfects hepatocytes in the intact animal. These results suggest that transfection of hepatocytes with ADV-D1 can promote hepatocyte proliferation independent of extracellular signals, and warrants testing as an agent to promote liver regeneration. The three Specific Aims of this study will examine the mechanisms by which cyclin D1 promotes hepatocyte growth, and will examine the ability of other G1 phase cyclins and E2F transcription factors to promote hepatocyte cell cycle progression. These studies are intended to provide unique mechanistic insight in the regulation of hepatocyte growth, and to identify candidate proteins for gene targeting strategies to therapeutically enhance liver regeneration.