ESTABLISHMENT OF HUMAN LENS EPITHELIAL CELL LINES

白内障研究的目的是预防或延缓人类白内障的形成。白内障是世界上主要的致盲原因。上皮功能受损必然导致透明度的丧失。一种旨在延缓或预防人类白内障的疗法需要在人类晶状体上皮细胞(HLECs)上进行评估。由于人类晶状体的稀缺，许多研究人员试图培养人晶状体上皮细胞。然而，正常情况下，HLECs在培养过程中完成2-4代并经历衰老。HLECs具有不同于低等物种的特性。例如，可以使用标准技术从低等物种建立晶状体上皮系；但HLEC并非如此。相对于多元醇途径，不同物种的代谢通量也不同。醛糖还原酶活性在狗和大鼠的晶状体中很高，在人类晶状体中很低，在小鼠中不存在。尽管HLECs的病毒转化允许生长，但病毒转化的细胞具有与未转化细胞不同的特性。需要通过实验来确定正常HLECs的性质。我们的目标是建立正常的HLEC的可靠供应，对它们进行表征，并将它们提供给眼科研究社区。这对今后的研究是绝对必要的。其目的是：1.确定伤口愈合环境是否允许来自年轻、老年和白内障晶状体的正常HLEC长期生长。2.确定将端粒酶导入HLECs是否允许从晶状体外植体建立系，是否允许已建立的系克服衰老，以及端粒酶的存在是否保持二倍体状态并允许体内特征保持。确定目标1或2产生的细胞的倍性、同工酶表型、DNA指纹、αA和αB晶状体蛋白的表达以及生长情况。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.在编码γ-谷氨酰半胱氨酸合成酶(GCS)的基因启动子中确定晶状体特异的调控元件。

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).