AGE & HORMONE DEPENDENT REGULATION OF HEPATIC PROTEIN

拟议研究的长期目标是更好地了解 衰老过程的分子机制及其与 改变内分泌环境。 这一目标将通过以下途径实现： 对年龄和年龄依赖性肝合成的调节研究 蛋白（SMP-2）。 SMP-2已被鉴定和表征为 激素作用和衰老的生物标志物。 拟议的研究将 强调以下几点：1）全长的分离和表征 使用表达载体的SMP-2的两种同种型的SMP-2 cDNA克隆; 2） SMP-2基因组克隆的分离和表征; 3） 鉴定可能显示高亲和力的核非组蛋白蛋白 SMP-2基因调控区的序列分析; 4）SMP-2基因的鉴定 通过免疫组织化学方法合成肝细胞， 探索SMP-2合成中可能的细胞特化; 5） 生产针对两种形式SMP-2的单克隆抗体; 6） 阐明SMP-2及其mRNA的雄激素阻遏机制 使用核径流分析和脉冲追踪研究等实验 用于分析mRNA稳定性; 7）探索可能的组织多样性 在使用RNA斑点印迹分析的SMP-2的合成中， 免疫组化;和8）调查的时间过程中， SMP-2的再现在老年，在热量限制的大鼠， 测量SMP-2 mRNA水平。

The long-term goal of the proposed research is a better understanding of the molecular mechanism of the aging process and its relationship to changing endocrine environment. This aim will be pursued through the regulatory studies on the age- and hormone-dependent synthesis of a hepatic protein (SMP-2). SMP-2 has been identified and characterized as a biomarker for hormone action and aging. The proposed research will emphasize the following: 1) isolation and characterization of full length SMP-2 cDNA clones for two isoforms of SMP-2 using an expression vector; 2) isolation and characterization of the genomic clones for SMP-2; 3) identification of nuclear nonhistone proteins that may show high affinity for the regulatory region of SMP-2 gene; 4) identification of the SMP-2 synthesizing hepatocytes by the immunohistochemical method in order to explore possible cellular specialization in the synthesis of SMP-2; 5) producation of monoclonal antibodies to the both forms of SMP-2; 6) elucidation of the mechanism of androgenic repression of SMP-2 and its mRNA using such experiments as nuclear run off assays, and pulse-chase studies for analysis of mRNA stability; 7) exploration of possible tissue diversity in the synthesis of SMP-2 using RNA dot blot assay and immunohistochemistry; and 8) investigation of the time course of reappearance of SMP-2 at the old age, in caloric restricted rats, by measuring the SMP-2 mRNA level.