PILOT STUDY--PI 3'-KINASE AND HEPATOCYTE PROLIFERATION

部分肝损伤后肝细胞再生的主要介质 肝切除术是一种肝细胞生长因子，其受体是一种 膜酪氨酸激酶已知可激活脂蛋白激酶 HGF刺激的细胞内的磷脂酰肌醇3‘-激酶(PI 3’-Kinase)。 这项先导性和可行性研究将检验PI3‘-激酶的作用 在肝细胞增殖过程中，使用最小偏差率 以粮农组织为模型系统的肝癌株系。粮农组织的细胞保留了许多肝脏- 具体功能。此外，粮农组织的细胞可以从静止的 通过在细胞中添加胰岛素而进入增殖状态 缺乏血清或其他生长因子。作为与HGF共享的胰岛素 激活PI 3‘-激酶的能力，这些细胞提供了一个有用的 研究肝细胞调节的模型系统 扩散。这项建议中使用的方法是调整 导入PI3‘-激酶在FAO细胞中的活性 激活的或显性的负性突变；胰岛素的变化- 被刺激的PI 3‘-激酶活性将与 胰岛素诱导的有丝分裂反应的敏感性。激活的交点 3‘-激酶突变体将通过引入序列来引导 脂质部分的添加，从而导致结构性 PI 3‘-激酶与细胞内膜的结合。占优势 负突变体将由突变的残基产生 PI 3‘-激酶的调节和催化亚基是同源的 到相关蛋白质中的关键残基。这些因素的影响 对胰岛素刺激的增殖的扰动，以及胰岛素 对肝脏特异性代谢酶的刺激，将被确定。 这些研究应该是对PI作用的有价值的初步探讨 肝细胞生理学中的3‘-激酶。

A major mediator of hepatocyte regeneration following partial hepatectomy is Hepatocyte Growth Factor (HGF), whose receptor is a membrane tyrosine kinase known to activate the lipid kinase phosphatidylinositol 3'-kinase (PI 3'-kinase) in HGF-stimulated cells. This Pilot and Feasibility study will examine the role of PI 3'-kinase during hepatocyte proliferation, using, the minimal deviation rat hepatoma line Fao as a model system. Fao cells retain many liver- specific functions. Moreover, Fao cells can be moved from a quiescent state into a proliferative state by the addition of insulin in the absence of serum or other growth factors. As insulin shares with HGF the ability to activate PI 3'-kinase, these cell provide a useful model system in which to study the regulation of hepatocyte proliferation. The approach use in this proposal is to modulate the activity of PI 3'-kinase in Fao cells through the introduction of activated or dominant negative mutants; alterations in insulin- stimulated PI 3'-kinase activity will be correlated with changes in the sensitivity of insulin-induced mitogenic responses. Activated PI 3'-kinase mutants will be created by introducing sequences that direct the addition of lipid moieties, thereby causing the constitutive association of PI 3'-kinase with intracellular membranes. Dominant negative mutants will be created by mutating residues in the regulatory and catalytic subunits of PI 3'-kinase that are homologous to critical residues in related proteins. The effect of these perturbations on insulin-stimulated proliferation, as well as insulin stimulation of liver-specific metabolic enzymes, will be determined. These studies should be a valuable initial inquiry into the role of PI 3'-kinase in hepatocyte physiology.