Promoting Corneal Graft by Anti-Apoptotic Genes

这是一项探索性/发展性拨款申请，旨在探索细胞凋亡在角膜中的作用。 移植失败。尽管使用了免疫抑制，但许多角膜移植物无法存活，数据表明，角膜移植后很长一段时间内，移植物内皮细胞会逐渐丢失。我们的初步数据为将基因导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，过表达抗凋亡因子Bcl-xl的转移者的角膜移植的存活率显著提高，这导致了我们的总体假设，即移植物中细胞凋亡的下调可以提高移植物的存活率。基于这一假设，我们确定了两个目标：特定的目标1.检测过表达抗凋亡基因对体外培养的角膜内皮细胞(CEC)存活和角膜移植物存活的影响 活着。在体外研究中，我们将过表达感兴趣的抗凋亡基因(Bcl2、Bclxl和p35)，并测试在不同类型的凋亡刺激(H202、细胞因子和DNA损伤剂)下过表达对CEC存活的影响。体内研究将通过在标准验证的原位角膜移植模型中测试转基因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(S)。特定目的2.检测抗细胞凋亡的基因治疗 基因可以减少小鼠角膜移植的失败。 综上所述，这些目标将在以下方面提供重要信息：1)移植细胞凋亡在影响移植存活中的总体作用；2)几种有效的抗凋亡因子对角膜内皮细胞存活的不同影响；3)可行的抗凋亡基因治疗干预措施，可以促进理想基因的表达进入角膜组织。该项目的长期目标是确定最终可能被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，仅在美国就有近4万例患者进行了移植。我们相信，我们的目标是新颖、创新的，非常适合我们实验室的技能，并描绘出细胞凋亡在角膜移植物存活中的作用。

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.