ESTABLISHMENT OF HUMAN LENS EPITHELIAL CELL LINES

人晶状体上皮细胞系的建立

• 批准号: 6165727
• 负责人: John R. Reddan
• 金额: $ 21.3万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6165727
• 关键词: age difference; cataract; cell growth regulation; cell line; crystallins; electroporation; enzyme activity; epithelium; genetic promoter element; genetic regulatory element; human tissue; isozymes; lens; organ culture; phenotype; polymerase chain reaction; restriction fragment length polymorphism; telomerase; tissue /cell preparation; transfection; western blottings

The aim of cataract research is to prevent or delay human cataract formation. Cataract is the world's leading cause of blindness. Impaired epithelial function invariably leads to a loss of transparency. A therapy aimed at delaying or preventing human cataracts needs to be evaluated on human lens epithelial cells (HLECs). Due to the scarcity of viable human lenses, numerous investigators have tried to culture HLECs. However, normal, HLECs complete 2-4 passages in culture and undergo senescence. HLECs have properties different from those of the lower species. For example, lens epithelial lines can be established from lower species using standard techniques; this is not true of HLECs. Metabolic fluxes in various species are also different relative to the polyol pathway. Aldose reductase activity is high in the dog and rat lens, very low in the human lens and absent in the mouse. Although viral transformation of HLECs permits growth, virally transformed cells have properties that are different from their non-transformed counterparts. Experiments are needed to determine the properties of normal HLECs. Our aims are to establish a reliable supply of normal HLECs, characterize them and make them available to the eye research community. This is absolutely necessary for future studies. The aims are to: 1. Determine if a wound-healing environment permits the long-term growth of normal HLECs from young, old and cataractous lenses. 2. Determine if the introduction of telomerase into HLECs permits establishment of lines fromlens explants, permits established lines to overcome senescence, and if the presence of telomerase maintains the diploid state and allows maintenance of in vivo characteristics. Determine the ploidy, isoenzyme phenotyping, DNA fingerprint, alphaA and alphaB crystallin expression, and growth in cells generated Aim 1 or 2. 3. Determine if there are metabolic differences between cells from young, old and cataractous lenses. 4. Identify lens-specific regulatory elements in the promoters of genes that encode gamma-glutamylcysteine synthetase (GCS).

白内障研究的目的是预防或延缓人类白内障的形成。 白内障是世界上导致失明的主要原因。 受损的上皮功能总是导致透明度的丧失。 需要在人透镜上皮细胞（HLEC）上评估旨在延迟或预防人白内障的疗法。 由于缺乏可行的人类晶状体，许多研究人员试图培养HLEC。 然而，正常的HLEC在培养中完成2-4次传代并经历衰老。 HLEC具有不同于低等物种的性质。 例如，透镜上皮细胞系可以使用标准技术从低等物种建立;这对于HLEC是不正确的。 不同物种的代谢通量相对于多元醇途径也不同。 在犬和大鼠透镜中，醛糖还原酶活性较高，在人透镜中极低，在小鼠中不存在。 虽然HLEC的病毒转化允许生长，但病毒转化的细胞具有不同于其非转化对应物的性质。需要实验来确定正常HLEC的性质。 我们的目标是建立正常HLEC的可靠供应，表征它们并使其可用于眼科研究界。 这对今后的研究是绝对必要的。 目的是：1.确定伤口愈合环境是否允许年轻、老年和白内障晶状体的正常HLEC长期生长。2.确定是否将端粒酶引入HLEC允许从晶状体外植体建立细胞系，允许建立的细胞系克服衰老，以及端粒酶的存在是否维持二倍体状态并允许维持体内特征。 确定Aim 1或2产生的细胞的倍性、同工酶表型、DNA指纹、α A和α B晶体蛋白表达以及生长。3.确定年轻、老年和白内障晶状体细胞之间是否存在代谢差异。4.识别编码γ-谷氨酰半胱氨酸合成酶（GCS）的基因启动子中的晶状体特异性调控元件。