MOLECULAR ANALY MICRODISSECTED CATARACTOUS HUMAN LENSES

这项提案的长期目标是提供有关年龄相关性白内障的保护和调节基因的身份和功能的知识。 该建议的中心前提是，透镜上皮细胞对于维持透镜的正常功能是必需的，并且透镜上皮细胞的功能组分响应于在白内障发生期间诱导的生理变化。 本研究的具体目的是：（1）鉴定人透镜上皮细胞中在年龄相关性白内障中表达上调或下调的基因，并评估这些基因在透镜中的潜在功能。 (2)确认和表征从白内障和正常晶状体解剖的人透镜上皮细胞之间鉴定的基因的mRNA水平、蛋白水平和空间表达模式;确定鉴定的基因在特定类型白内障中的表达水平;以及确定这些基因的表达水平与其在透镜中的潜在功能之间的关系。 这些目标将通过整合的方法来实现，包括逆转录酶差异显示，序列分析，克隆新的差异表达基因，半定量RT-PCR，Western分析，原位杂交和免疫组织化学。 这种方法的可行性证明了六个基因的初步鉴定，这是向上或向下调节，在响应年龄相关性白内障和表征和分析这些基因中的三个在正常和白内障的人类晶状体。 随后报道了这些基因中的一种，称为骨粘连蛋白或骨粘连蛋白（Gilmour et al.，EMBO J.，1998）1在小鼠中缺失时引起老年性白内障形成，进一步证明了本研究中概述的方法的有用性，并证明了该提议中鉴定的基因在其正常透镜功能丧失时也可能是引起白内障的候选基因。 从这里提出的工作中获得的信息将被用来连接白内障特异性基因表达的变化与特定的透镜的功能，重要的是保持透镜的透明度，并应提供一个合理的基础，朝着发展的方法，以延缓年龄相关性白内障。

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.