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以下因素诱导： 大量的代理商。 然而，共同的细胞机制， 这些不同的药物引起白内障仍然是未知的。 我们最近 正常人和正常人的透镜上皮细胞活力的研究 白内障晶状体和有或无氧化损伤的大鼠晶状体 应力、卡西霉素和UVB一致地表明诱导透镜 上皮细胞凋亡是这样一种常见的细胞机制。 因此，我们认为， 是否抑制应激诱导的透镜上皮细胞凋亡， 成熟的透镜在各种应激下可延缓或预防白内障的发生 环境成为关系到人类健康的重要问题， 获得这个问题的答案肯定会有助于了解 人类白内障的潜在基因治疗。 由于细胞凋亡一直是 据报道，在大鼠透镜的正常发育中发生，此外， 透镜纤维细胞的分化过程（包括初级和次级 纤维）在发展和成熟的晶状体共享某些共同的途径 在凋亡过程中，任何试图延迟或阻止 通过简单地抑制发育中的透镜中的细胞凋亡而导致的白内障发生可能 干扰透镜的正常显影。 因此，建议 在这里进行正常生理性细胞凋亡的比较研究， 初生纤维细胞和次生纤维细胞发育过程中的透镜、固缩 发育和成熟晶状体的分化以及应力诱导的 大鼠、小鼠和鸡成熟晶状体的病理性凋亡 从基因表达和信号转导两个方面进行研究。 到 分析基因表达，逆转录连接聚合酶链 反应和原位杂交将用于确定是否存在 或不存在所选凋亡调节基因的mRNA（Rb， p53，c-myc，c-jun，c-fos，p34cdc2，bcl-2，bcl-x，bax，bad，bag-1，mch-1， 冰和ICH-1）。 特定基因的mRNA表达的存在 将通过免疫细胞化学分析进一步证实， 蛋白质的表达。 进行信号传导 研究，RNA和蛋白质合成抑制剂的作用， 各种激酶和磷酸酶抑制剂，以及激酶激活剂， 以上三个过程将通过离体培养进行验证 然后研究他们的反应。 这些研究将阐明 三个过程的异同，并帮助确定 是否有一个特定的实验策略来抑制压力， 诱导成熟透镜中的上皮细胞凋亡，同时不干扰 具有正常的透镜生长和分化。 然后，研究确定， 抑制应激诱导的细胞凋亡是否可以延缓或阻止 成熟晶状体的白内障发生将在体外进行， 透镜器官培养系统。 与此同时， 一组6个基因：p35，crmA，bcl-xL，bag-1，mcl-1和ich-IS已知 将在体外细胞中测试抑制非晶状体系统中的细胞凋亡 株系，N/N1003 A和alphaTN 4 -1。 在确认他们的 抑制应激诱导的细胞凋亡的能力，阳性基因将 用于开发过表达转基因模型。 表型（例如， 这些转基因小鼠的透镜透明度和抗细胞凋亡能力）将 与正常小鼠进行比较。 结果从这些 研究不仅为未来的设计提供了宝贵的信息， 基因治疗对人类白内障的预防，也增加了我们的 了解正常透镜的发育生物学。

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.