LENS EPITHELIUM AND HELIUM ION CATARACTOGENESIS

晶状体上皮和氦离子白内障发生

• 批准号: 2518763
• 负责人: ELEANOR A BLAKELY
• 金额: $ 29.55万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2518763
• 关键词: DNA damage; HeLa cells; SDS polyacrylamide gel electrophoresis; cataract; cell differentiation; cellular pathology; chromosome aberrations; cytogenetics; enzyme linked immunosorbent assay; eye neoplasms; fibroblast growth factor; helium; human tissue; iatrogenic disease; in situ hybridization; lens proteins; melanoma; messenger RNA; neoplasm /cancer radiation therapy; northern blottings; radiation genetics; radiation therapy dosage; tissue /cell culture; tissue mosaicism; western blottings

Cataracts arise in uveal melanoma patients as a complication following their successful treatment with helium-ion radiotherapy. The objective of the proposed research is to determine the helium-ion-induced alterations in chromosomes and in protein expression that are important to cataractogenesis, and to develop strategies to diminish the incidence or severity of these changes. The current paradigm for cataractogenesis is focussed on genomic damage of lens epithelial cells leading to altered crystallin proteins. This proposal will examine two alternative mechanisms of cataractogenesis involving radiation-induction of basic Fibroblast Growth Factor (bFGF) in human lens epithelial (HLE) cells functioning either to alter the normal program of crystallin expression and thereby disrupting normal fiber formation, or the radiation-induced bFGF acting to hinder cell loss processes which leads to the formation of aberrant lens fiber formation. To this end experiments have been designed with three specific aims that will: Firstly, characterize acute and residual responses to helium-ion irradiation of cultured HLE cells grown on extracellular matrix. Quantitative dose-response measurements between helium-ion induced survival, repair capacity and yields of micronuclei, apoptotic nuclei, and persistent chromosomal rearrangements will be determined. Secondly, since radiation is known to induce bFGF in endothelial cells leading to inhibition of apoptosis, experiments are proposed that will determine whether helium-ion radiation changes levels of bFGF mRNA or protein in HLE cells, and whether there is a correlation with apoptotic events. Finally, an investigation of the possible modification of intracellular lens proteins by helium-ion radiation is proposed. The program will elucidate relationships between helium-ion- induced damage to the chromatin of HLE cells in vitro, and biological consequences to the cells surviving the resulting damage. In the revised application significant changes include: 1) an increased source of HLE cells, 2) new preliminary data from mRNA and protein analyses (SDS-PAGE) of exponential and confluent cell cultures, 3) new preliminary data with immunological evidence confirming feasibility to characterize lens cell differentiation, 4) some revised experimental protocols, and 5) and an updated review of literature published in this rapidly increasing area. This knowledge will allow correlative comparisons with available experimental work in vivo, and may provide a basis for potential changes to the treatment protocol that could reduce the risk of cataract. The biological mechanisms contributing to cataractogenesis may also be elucidated.

白内障是葡萄膜黑色素瘤患者出现以下并发症的一种情况： 他们通过氦离子放射治疗取得了成功。目标 拟议研究的目的是确定氦离子引起的 重要的染色体和蛋白质表达的改变 白内障的发生，并制定减少其发病率的策略 或这些变化的严重程度。当前白内障发生的范例 专注于晶状体上皮细胞的基因组损伤导致改变 晶状体蛋白。该提案将研究两种替代方案 白内障发生机制涉及基础辐射诱导 人晶状体上皮 (HLE) 细胞中的成纤维细胞生长因子 (bFGF) 功能要么改变晶状体蛋白表达的正常程序 从而破坏正常的纤维形成，或辐射引起的 bFGF 会阻碍细胞丢失过程，从而导致形成 异常晶状体纤维的形成。为此进行了实验 设计的三个具体目标是：首先，刻画尖锐的特征 培养的 HLE 细胞对氦离子照射的残余反应 生长在细胞外基质上。定量剂量反应测量 氦离子诱导的存活、修复能力和产量之间 微核、凋亡核和持续染色体重排 将被确定。其次，由于已知辐射会诱导 bFGF 内皮细胞导致细胞凋亡的抑制，实验是 提议将确定氦离子辐射是否会改变水平 HLE 细胞中 bFGF mRNA 或蛋白的变化，以及是否存在相关性 与凋亡事件。最后，对可能的情况进行调查 氦离子辐射对细胞内晶状体蛋白的修饰是 建议的。该计划将阐明氦离子之间的关系 体外诱导 HLE 细胞染色质损伤，并进行生物学研究 对在所造成的损伤中幸存下来的细胞造成的后果。在修订后的 应用重大变化包括： 1) HLE 来源增加 细胞，2) 来自 mRNA 和蛋白质分析 (SDS-PAGE) 的新初步数据 指数和汇合细胞培养物，3）新的初步数据 免疫学证据证实表征晶状体细胞的可行性 差异化，4）一些修订的实验方案，以及 5）和 在这个快速增长的领域发表的文献的最新评论。 这些知识将允许与可用的相关比较 体内实验工作，并可能为潜在的变化提供基础 可以降低白内障风险的治疗方案。这 导致白内障发生的生物学机制也可能是 阐明了。