Promoting Corneal Graft by Anti-Apoptotic Genes

这是一个探索性/发展资助申请，旨在探索细胞凋亡在角膜上皮细胞中的作用。 移植失败尽管使用免疫抑制，许多角膜移植物不能存活，并且数据表明，角膜移植后很长一段时间内移植物内皮细胞逐渐丧失。我们的初步数据为将基因引入角膜内皮和基质的可行性提供了支持。此外，我们有数据表明，过度表达抗凋亡因子Bcl-xL的转基因角膜移植物具有显著提高的存活率，这导致我们的总体假设，即移植物中凋亡的下调可以促进移植物存活。基于这一假设，我们确定了两个目标：具体目标1。测试过表达抗凋亡基因对a）体外培养的角膜内皮细胞（CEC）的存活和B）角膜移植物的存活的影响。 vivo.对于体外研究，我们将过表达感兴趣的抗凋亡基因（Bcl-2、Bcl-xL和p35），并测试这种过表达对暴露于不同类型的凋亡刺激物（H2 O2、细胞因子和DNA损伤剂）后CEC存活的影响。体内研究将通过在标准的经验证的原位角膜移植模型中测试转基因动物中这些因子的选择性过表达对移植物存活的影响来补充体外研究。来自特定目标1的数据将有助于确定与促进角膜移植物存活相关的最佳基因。具体目标2。检测抗凋亡基因治疗是否 基因减少小鼠角膜移植失败。 总之，这些目标将提供以下方面的重要信息：1）移植细胞凋亡在影响移植存活中的总体作用，2）几种有效抗凋亡因子对角膜内皮细胞存活的差异作用，3）可促进所需基因在角膜组织中表达的可行抗凋亡基因治疗干预。该项目的长期目标是确定最终可能用于角膜移植的抗凋亡策略，角膜移植是最常见的组织移植形式，仅在美国每年就有近40，000例。我们相信，我们的目标是新颖的，创新的，非常适合我们的实验室的技能，并在描绘角膜移植存活的细胞凋亡的作用。

This is an exploratory/development grant application to explore the role of apoptosis in corneal transplant failure. In spite of the use of immune suppression, many corneal grafts do not survive, and data suggest that graft endothelial cells are progressively lost for a long time after corneal transplantation. Our preliminary data provide support for the feasibility of introducing genes into both the corneal endothelium and stroma. Additionally, we have data suggesting that corneal transplants from transgenics that overexpress the anti-apoptotic factor Bcl-xL have a significantly enhanced survival rate, leading to our overarching hypothesis that downmodulation of apoptosis in the graft can promote graft survival. Based on this hypothesis, we have identified two aims: Specific Aim 1. Test the effect of overexpressing anti-apoptotic genes on a) the survival of cultured corneal endothelial cells (CEC) in vitro and b) survival of corneal grafts in vivo. For the in vitro studies, we will overexpress anti-apoptotic genes of interest (Bcl-2, Bcl-xL, and p35) and test the effect of such overexpression on survival of CEC following exposure to different types of apoptotic stimuli (H202, cytokines, and DNA damaging agents). The in vivo studies will complement the in vitro studies by testing the effect of select overexpression of these factors in transgenics on graft survival in a standard validated model of orthotopic corneal transplantation. Data from Specific Aim 1 will help identify the optimal gene(s) relevant for promoting corneal graft survival. Specific Aim 2. Test whether gene therapy with antiapoptosis genes reduces failure of corneal grafts in mice. Taken together, these aims will provide significant information on 1) overall role of graft cell apoptosis in affecting transplant survival, 2) differential effect of several potent anti-apoptotic factors on survival of corneal endothelial cells, and 3) feasible anti-apoptotic gene therapy interventions that can promote expression of desirable genes into corneal tissues. The long-term objective of this project is to identify anti-apoptotic strategies that may eventually be adopted for use in corneal transplantation, the most common form of tissue grafting, with nearly 40,000 cases performed annually in the United States alone. We believe that our goals are novel, innovative, and highly suited to the skills of our laboratory, and in delineating the role of apoptosis in corneal graft survival.