APOPTOSIS--LENS DIFFERENTIATION AND CATARACT DEVELOPMENT

已经有充分的文献证明，白内障的发生可以由一种 大量的代理。然而，常见的细胞机制 这些不同的因素导致白内障的原因至今仍不清楚。我们最近 正常人和正常人晶状体上皮细胞活性的研究 白内障晶状体和氧化损伤前后的大鼠晶状体 应激、钙霉素和UVB一致表明诱导的晶状体 上皮细胞凋亡是一种常见的细胞机制。因此， 抑制应激诱导的晶状体上皮细胞凋亡 成熟晶状体在不同的压力下可以延缓或防止白内障的发生。 环境成为关系到人类健康的重要问题 得到这个问题的答案肯定会对 人类白内障的潜在基因治疗。因为细胞凋亡已经 据报道发生在大鼠晶状体的正常发育中，此外， 晶状体纤维细胞(原代和次级)的分化过程 纤维)在发育和成熟的晶状体中共享某些共同的途径 在细胞凋亡过程中，任何试图推迟或阻止 通过简单地抑制发育中的晶状体中的细胞凋亡来实现白内障的发生可能 干扰晶状体的正常发育。因此，建议 在此对正常生理性细胞凋亡进行比较研究 初级纤维细胞和次级纤维细胞发育中的晶状体固缩 发育成熟晶状体的分化与应激诱导 大鼠、小鼠和鸡成熟晶状体中的病理性细胞凋亡 包括基因表达和信号转导两个方面。至 分析基因表达，反转录连接聚合酶链 将使用反应和原位杂交来确定是否存在 或不存在所选的凋亡调节基因(Rb， P53、c-myc、c-jun、c-fos、p34cdc2、bcl2、bclx、bax、BAD、Bag-1、Mch-1、 ICE和ICH-1)。存在特定基因的信使核糖核酸表达 将通过免疫细胞化学分析进一步证实 蛋白质的表达。进行信号转导 研究，RNA和蛋白质合成抑制剂的效果， 多种激酶和磷酸酶抑制剂，以及一种 上述三个过程将通过体外培养进行检测。 然后是对他们的反应的研究。这些研究将阐明 这三个过程的异同和帮助确定 是否有一种特定的实验策略来抑制压力- 不干预诱导成熟晶状体上皮细胞凋亡 晶状体生长和分化正常。然后，研究确定 抑制应激诱导的细胞凋亡能否延缓或预防 成熟晶状体的白内障形成将在体外进行。 晶状体器官培养系统。同时，抗细胞凋亡的能力 在一组6个基因中：p35、crmA、bclxl、Bag-1、mcl1和ich-已知 在非晶状体系统中抑制细胞凋亡将在体外细胞中进行测试 品系N/N1003A和αTn4-1。在确认了他们的 能够抑制应激诱导的细胞凋亡，阳性基因会 用于开发过度表达的转基因模型。表型(例如， 这些转基因小鼠的晶状体透明性和抗凋亡能力)将 以正常小鼠为对照进行检查。这些研究的结果 研究不仅将为未来的设计提供有价值的信息 基因疗法预防人类白内障，但也增加了我们的 了解正常晶状体的发育生物学。

It has been well documented that cataractogenesis can be induced by a large number of agents. However, the common cellular mechanism by which these different agents cause cataract has remained unknown. Our recent studies of the lens epithelial cell viability in human normal and cataractous lenses and in rat lenses with or without insult by oxidative stress, calcimycin and UVB consistently suggest that induced lens epithelial cell apoptosis is such a common cellular mechanism. Therefore, whether inhibition of the stress-induced lens epithelial apoptosis in the mature lens can retard or prevent cataractogenesis under various stress environments becomes an important question related to human health and obtaining the answer to this question will certainly shed some light to the potential gene therapy for human cataract. Since apoptosis has been reported to occur in normal development of the rat lens and moreover, the differentiation process of lens fiber cells (both primary and secondary fibers) in the developing and mature lenses share certain common pathways with the apoptotic process, any attempt to delay or prevent cataractogenesis by simply inhibiting apoptosis in the developing lens may interfere with normal development of the lens. Therefore it is proposed here to conduct comparative studies of normal physiological apoptosis in the developing lens, pycnosis during both primary and secondary fiber cell differentiation in the developing and mature lenses and stress-induced pathological apoptosis in the mature lenses of rat, mouse and chicken with respect to two aspects: gene expression and signal transduction. To analyze gene expression, reverse-transcription-linked polymerase chain reaction and in situ hybridization will be used to determine the presence or absence of the mRNAs for the selected apoptosis-regulatory genes (Rb, p53, c-myc, c-jun, c-fos, p34cdc2, bcl-2, bcl-x, bax, bad, bag-1, mch-1, ice and ich-1). The presence of the mRNA expression for a particular gene will be further confirmed by immunocytochemistry analysis which detects the expression of the protein. To conduct signal transduction investigations, the effects of both RNA and protein synthesis inhibitors, various kinase and phosphatase inhibitors, and a kinase activator on the above mentioned three processes will be examined by in vitro culture followed by studies of their responses. These studies will elucidate the similarities and differences of the three processes and help to determine whether there is a specific experimental strategy to inhibit only stress- induced epithelial cell apoptosis in the mature lens while not interfering with normal lens growth and differentiation. Then, studies to determine whether inhibition of the stress-induced apoptosis could delay or prevent cataractogenesis in the mature lenses will be conducted with the in vitro lens organ culture systems. At the same time, the antiapoptotic abilities of a group of 6 genes: p35, crmA, bcl-xL, bag-1, mcl-1 and ich-IS known to inhibit apoptosis in non-lens systems will be tested in the in vitro cell lines, N/N1003A and alphaTN4-1. Following the confirmation of their ability to suppress stress-induced apoptosis, the positive genes will be used to develop over-expression transgenic models. The phenotype (e.g., lens transparency and antiapoptotic ability) of these transgenic mice will be examined with comparison to the normal mice. The results from these studies will not only provide valuable information for future design of gene therapy for human cataract prevention but also increase our understanding of the developmental biology of the normal lens.