AGE & HORMONE DEPENDENT REGULATION OF HEPATIC PROTEIN

拟议研究的长期目标是更好地理解 衰老过程的分子机制及其与衰老的关系 不断变化的内分泌环境。这一目标将通过 一种依赖年龄和激素的肝脏合成的调控研究 蛋白质(SMP-2)。SMP-2已被鉴定为一种 荷尔蒙作用和衰老的生物标志物。拟议的研究将 强调以下几点：1)全长的分离和鉴定 利用表达载体克隆SMP-2两种异构体的cDNA； SMP-2基因克隆的分离与鉴定 可能显示高亲和力的核非组蛋白的鉴定 SMP-2基因调控区；4)SMP-2基因的鉴定 用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) 制备抗SMP-2两种形式的单抗；6) SMP-2及其mRNA雄激素抑制机制的阐明 利用核径流分析和脉冲追逐研究等实验 用于分析mRNA的稳定性；7)探索可能的组织多样性 在SMP-2合成中的RNA斑点印迹分析和 免疫组织化学；8)时间进程的研究。 SMP-2在老年热量限制大鼠中的重新出现，通过 检测SMP-2mRNA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.