GROWTH CONTROL IN AGING FIBROBLASTS

该提案旨在了解控制 在成纤维细胞增殖的最后阶段终止细胞复制 寿命;我们假设一系列基因的激活， 其产物直接或间接起抑制剂的作用， 使非增殖性衰老成纤维细胞从 穿越细胞周期。 为此，我们已经确定了他汀类，一种蛋白质表达 仅在非增殖细胞中，并被表征为 G 0-静止细胞。 在本提案中，我们计划：（1）表征 他汀类药物的生物化学合成、修饰和降解; (2)纯化天然和变性形式的他汀类药物， 生物化学分析、蛋白质测序和抗体生产; (3)分析他汀类药物如何与细胞核的成分相互作用， （4）他汀基因的克隆和鉴定;（5）分析 他汀类药物的细胞功能;（6）分析其与细胞凋亡的关系 在体外和体内老化之间，使用他汀作为探针;和（7） 鉴定其他缎样蛋白。 蛋白质定位的结构方面将通过 光镜和电镜免疫标记技术 程度. 生物化学研究将通过细胞分级进行 随后进行1-D和2-D凝胶分析， 免疫沉淀和免疫印迹分析。 蛋白质将 通过电泳分离纯化，各种柱 色谱法和HPLC。 分子克隆将是 通过构建和筛选含他汀的c- DNA文库 功能研究将由 用单特异性抗体显微注射单细胞， 纯化的蛋白质和c-DNA克隆。 组织化学检查 在来自不同的动物组织中的纤维细胞中， 年龄组将开始尝试在体外 成纤维细胞老化到体内器官老化。 其他他汀类药物 蛋白质将通过产生独特的单克隆抗体来鉴定。 抗体能够识别罕见的，不扩散的， 依赖蛋白质 我们的研究结果应该从 现象学观察 细胞衰老的研究。 单克隆抗体等工具 抗体，纯化的蛋白质和c-DNA克隆，在这方面的发展， 工作，将创建一个强大的实验探针库， 不仅在衰老成纤维细胞的研究中， 增长一般。 这里提出的问题的答案不仅是 对细胞衰老至关重要，但对于理解 在发育、分化和肿瘤发生中的调节。

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.