MOLECULAR ANALY MICRODISSECTED CATARACTOUS HUMAN LENSES

本研究的长期目标是提供对老年性白内障的保护和调控基因的身份和功能的认识。该建议的中心前提是晶状体上皮对维持晶状体的正常功能至关重要，并且晶状体上皮的功能成分对白内障发生过程中诱导的生理变化有反应。本提案的具体目的是：(1)鉴定人类晶状体上皮中表达上调或下调的基因，以响应年龄相关的人类白内障，并评估这些基因在晶状体中的潜在功能。(2)确认并表征白内障晶状体上皮与正常晶状体上皮中鉴定基因的mRNA水平、蛋白水平及空间表达模式；目的：建立所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。这些目标将通过包括逆转录酶差异展示、序列分析、新型差异表达基因克隆、半定量RT-PCR、western分析、原位杂交和免疫组织化学在内的综合方法来实现。这种方法的可行性已被初步鉴定为六个基因，这些基因在年龄相关的白内障反应中被上调或下调，并在正常和白内障的人类晶状体中对其中三个基因进行了表征和分析。其中一种被称为骨连接素或SPARC的基因随后被报道（Gilmour等人，EMBO J., 1998）1，当在小鼠中缺失时，会导致老年性白内障形成，进一步证明了本研究中概述的方法的有效性，并证明在本建议中鉴定的基因也可能是当其正常晶状体功能丧失时导致白内障的候选基因。从本研究中获得的信息将用于将白内障特异性基因表达变化与维持晶状体透明度的特定晶状体功能联系起来，并为开发延缓年龄相关性白内障的方法提供合理的依据。

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.