MOLECULAR ANALY MICRODISSECTED CATARACTOUS HUMAN LENSES

这项建议的长期目标是提供关于保护和调节基因的身份和功能的知识，这些基因对年龄相关性白内障的存在做出反应。这一建议的中心前提是晶状体上皮细胞对维持晶状体的正常功能是必不可少的，晶状体上皮细胞的功能成分对白内障发生过程中诱导的生理变化做出反应。这一建议的具体目的是：(1)确定人类晶状体上皮细胞表达上调或下调的基因，以响应年龄相关性人类白内障，并评估这些基因在晶状体中的潜在功能。(2)确定并鉴定从白内障和正常晶状体中分离的人晶状体上皮细胞中已鉴定基因的mRNA水平、蛋白水平和空间表达模式；建立已鉴定基因在特定类型白内障中的表达水平；并建立这些基因的表达水平与其在晶状体中的潜在功能之间的关系。这些目标将通过综合方法实现，包括逆转录酶差异显示、序列分析、新的差异表达基因的克隆、半定量RT-PCR、蛋白质分析、原位杂交和免疫组织化学。通过初步鉴定年龄相关性白内障的6个上调或下调的基因，以及在正常人和白内障患者的晶状体中对其中3个基因的描述和分析，证明了该方法的可行性。其中一种名为Osteonectin或SPARC的基因后来被报道(Gilmour等人，EMBO J.，1998)1当被删除时会在小鼠中导致老年性白内障的形成，进一步证明了本研究中概述的方法的有效性，并证明了该提案中确定的基因也可能在其正常晶状体功能丧失时导致白内障。从这里提出的工作中获得的信息将被用来将白内障特异性基因表达的变化与特定的晶状体功能联系起来，这对维持晶状体的透明度非常重要，并应为开发延缓年龄相关性白内障的方法提供合理的基础。

The long term objective of this proposal is to provide knowledge about the identity and function of the protective and regulatory genes that respond to the presence of age-related cataract. The central premise of this proposal is that the lens epithelium is essential for maintaining the normal function of the lens and that functional components of the lens epithelium are responsive to physiological changes induced during cataractogenesis. The Specific Aims of this proposal are: (1) To identify genes of the human lens epithelium whose expression is up- or down-regulated in response to age-related human cataract and to evaluate the potential functions of these genes in the lens. (2) To confirm and to characterize the mRNA levels, protein levels and spatial expression patterns of the identified genes between human lens epithelia dissected from cataractous and normal lenses; To establish the expression levels of the identified genes in specific types of cataracts; and To establish a relationship between the expression levels of these genes and their potential functions in the lens. These aims will be accomplished using integrated approach involving reverse-transcriptase differential display, sequence analysis, cloning of novel differentially expressed genes, semi-quantitative RT-PCR, western analysis, in situ hybridization and immunohistochemistry. The feasibility of this approach is demonstrated by the initial identification of six genes which are up- or down-regulated in response to age-related cataract and the characterization and analysis of three of these genes in normal and cataractous human lenses. One of these genes, called osteonectin or SPARC, was subsequently reported (Gilmour et al., EMBO J., 1998)1 to cause age-onset cataract formation when deleted in mice, further demonstrating the usefulness of the approach outlined in the present study and demonstrating that the genes identified in this proposal may also be candidates for causing cataract when their normal lens function is lost. The information gained from the work proposed here will be used to link cataract-specific gene expression changes with specific lens functions important for the maintenance of lens transparency and should provide a rational basis towards developing methods to retard age-related cataract.