MANIPULATING HEPATOCYTE PROLIFERATION BY GENE TARGETING.

该R03方案的目的是检测细胞周期蛋白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。成熟肝细胞保留了干细胞的功能，并能在肝损伤后重新植入肝脏。以往的研究表明，细胞周期蛋白D1的上调在通过细胞周期的G1期调控肝细胞的进程中起着重要作用。为了测试细胞周期蛋白D1是否能够促进丝裂原非依赖性肝细胞的增殖，我们构建了编码该蛋白的复制缺陷型腺病毒(ADV-D1)。我们的初步研究表明，在缺乏生长因子的情况下，AdV-D1而不是控制腺病毒可以促进原代肝细胞的细胞周期进程，并诱导肝细胞在通常抑制生长的细胞外基质上增殖。体内试验研究表明，ADV-D1能有效地在完整动物体内转染肝细胞。这些结果表明，AdV-D_1基因可以促进肝细胞的增殖，而不依赖于细胞外信号，可以作为促进肝再生的一种试剂。这项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，并将检测其他G1期细胞周期蛋白和E2F转录因子促进肝细胞周期进程的能力。这些研究旨在为调节肝细胞生长提供独特的机制洞察力，并为基因靶向策略识别候选蛋白质以治疗增强肝再生。

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.