GROWTH CONTROL IN AGING FIBROBLASTS

这项提议旨在了解控制 成纤维细胞终末阶段细胞复制的终止 寿命；我们假设一系列基因的激活， 其产品直接或间接起到抑制剂的作用， 使非增殖性衰老成纤维细胞丧失能力 遍历细胞周期。 为此，我们确定了他汀类蛋白，一种表达的蛋白质 仅在非增殖细胞中，并被描述为 G0-静止细胞。在本提案中，我们计划：(1)描述 生物化学：他汀类药物如何合成、修饰和降解； (2)提纯天然和变性形式的他汀类药物 生物化学分析、蛋白质测序和抗体产生； (3)分析他汀类药物如何与核成分相互作用 (4)他汀类基因的克隆和鉴定；(5)分析 他汀类药物的细胞功能；(6)分析二者之间的关系 在体外和体内老化之间，使用他汀类药物作为探针；以及(7) 鉴定其他类似缎子的蛋白质。 蛋白质定位的结构方面将由 光镜和电子显微镜的免疫标记技术 级别。生物化学研究将通过细胞分离来进行 然后进行一维和二维凝胶分析，结合 免疫沉淀和免疫印迹分析。蛋白质将是 经电泳分离纯化，各种色谱柱 层析和高效液相色谱。分子克隆将是 通过构建和筛选含有他汀类药物的c- DNA文库。功能研究将由以下人员进行 用单特异性抗体显微注射单个细胞， 纯化蛋白和c-DNA克隆。组织化学检查 不同来源动物组织中成纤维细胞中他汀类药物的表达 年龄组将是体外联系的开始尝试 成纤维细胞老化到体内生物体老化。其他类他汀类药物 蛋白质将通过生产独特的单克隆来鉴定 能够识别罕见的、不扩散的抗体- 依赖蛋白质。 我们的研究结果应该会推动生物老年学从 对机械论的现象学观察 细胞老化的研究。诸如单克隆等工具 抗体，纯化的蛋白质和c-DNA克隆，在这个 工作，将创造出一批强大的实验探测器 不仅在衰老成纤维细胞的研究中，而且在细胞分析中也是如此 总体上是增长的。这里提出的问题的答案不仅是 细胞衰老的中心，但也对理解 发育、分化和肿瘤发生的调控。

This proposal aims to understand the mechanisms that control the termination of cell replication at the final stage of fibroblasts' life span; we hypothesize that activation of a series of genes, whose products function directly or indirectly as inhibitors, incapacitates the nonproliferating senescent fibroblasts from traversing the cell cycle. Towards this end we have identified statin, a protein expressed only in nonproliferating cells, and characterized as a marker for G0-quiescent cells. In this proposal, we plan to: (1) characterize biochemically how statin is synthesized, modified and degraded; (2) purify statin in native and denatured forms needed for biochemcial analysis, protein sequencing and antibody production; (3) analyze how statin interacts with components of the nuclear envelope; (4) clone and characterize the statin gene; (5) analyze the cellular functions of statin; (6) analyze the relationship between in vitro and in vivo aging, using statin as a probe; and (7) identify other satin-like proteins. Structural aspects of protein localization will be performed by immunolabelling techniques at both light and electron microscopic levels. Biochemcial studies will be pursued by cell fractionation followed by 1- and 2-D gel analysis, coupled with immunoprecipitation and immunoblotting assays. Proteins will be purified by electrophoretic separation, various column chromatographies, and HPLC. Molecular cloning will be performed by construction and screening of a statin-containing c- DNA library. Functional studies will be performed by microinjection of single cells with monospecific antibodies, purified proteins and c-DNA clones. Histochemcial examination of statin in fibrocytes in tissues derived from animals of differing age groups will be the starting attempt to relate in vitro fibroblast aging to in vivo organismic aging. Other statin-like proteins will be identified by production of unique monoclonal antibodies capable of recognizing rare, nonproliferation- dependent proteins. Results of our studies should advance biogerontology from phenomenological observation towards the mechanisitic investigation of cellular aging. Tools such as monoclonal antibodies, purified proteins and c-DNA clones, developed in this work, will create a bank of powerful experimental probes useful not only in studies of aging fibroblasts, but also in analysis of cell growth in general. Answers to questions raised here are not only central to cellular aging, but also crucial for the understanding of regulation in development, differentiation and oncogenesis.