Regulatory Role of CTL-O2/Bcl2 in Epithelial Homeostasis

描述（由申请人提供）：我们过去的研究表明，接触透镜（CTL）配戴显著影响角膜上皮（EH）的正常稳态控制，导致表面细胞脱落减少、基底细胞滞留延长和增殖减少。这些效应的净总和是产生以中央上皮角膜厚度降低为特征的“减慢”（EH）或“停滞”上皮层。与此同时，CTL相关的缺氧也会导致角膜表面铜绿假单胞菌（PA）受体上调，从而增加微生物性角膜炎（MK）的风险。基于这些发现，两个重要的不同的和相关的问题仍然存在：（1）什么样的CTL穿着时间表产生最低的风险MK和最少的干扰EH？(2)什么样的机制调节正常角膜EH以及CTL佩戴如何干扰它们？最近的研究结果表明，Bcl-2蛋白的表达和定位（细胞质，细胞核）可能构成了这些事件的一个关键的关系调节位点。为了检验这一假设，我们将使用兔体内CTL模型和兔/小鼠眼睑闭合的模拟CTL磨损模型，这将允许在没有炎症、损伤或使用抗有丝分裂剂的情况下实验性调节EH和PA结合。我们还将使用表现出Bcl-2过表达（转基因功能获得）、Bcl-2功能丧失的Bcl-2 KO和Bax KO小鼠的3种遗传改变小鼠（WT对照），以确定Bcl-2蛋白在EH和CTL诱导的PA结合中的调节作用。利用我们新开发的技术（体内共聚焦;激光共聚焦三重荧光成像）来评估体内和离体的EH，我们将追求5个具体目标：（1）确定每天或从头连续CTL佩戴是否在人类中产生MK或EH紊乱的最小风险;（2）Bcl-2的表达与MK的部位、EH和风险之间存在显著的相关性正常兔和小鼠中的PA结合（睁眼，长时间闭眼）;（3）建立与（2）中相同的长期相关性（1、2周）在接触透镜暴露的兔角膜中使用不同类型的测试CTL [刚性、软性、硅水凝胶（Sih）]和透镜-O2透射;（4）用Bcl-2功能、Bcl-2 KO和TG稳定性测定小鼠的高血压状态。（功能丧失）或促凋亡Bax KO（WT对照）以及EH和MK风险的相关变化（PA结合）至Bcl-2表达位置;（5）通过眼睑闭合调节TG和KO小鼠角膜中的角膜EH和PA结合水平，有/没有用可变O2、小鼠特异性测试CTL的延长的CTL暴露（1、3、7、14天），确定对Bcl-2表达的影响，并将PA与EH结合的变化与Bcl-2表达/位置相关联。

DESCRIPTION (provided by applicant): Our past research has shown that contact lens (CTL) wear significantly impacts the normal homeostatic control of the corneal epithelium (EH), leading to reduced surface cell shedding, prolonged basal cell retention and decreased proliferation. The net sum of these effects is to produce a "slowed down" (EH) or "stagnant" epithelial layer characterized by decreased central epithelial corneal thickness. At the same time, CTL-associated hypoxia also produces upregulation of P. aeruginosa (PA) receptors on the corneal surface, thus increasing the risk for microbial keratitis (MK). Based on these findings, two important distinct and related questions remain: (1) what CTL wearing schedule produces the LOWEST risk for MK and least disturbance of EH?; (2) what mechanism(s) regulate normal corneal EH and how does CTL wear perturb them? Recent results suggest that Bcl-2 protein(s) expression and location (cytoplasm, nucleus) may constitute a critical nexus regulatory site for these events. To test this hypothesis, we will use an in vivo CTL model in rabbits and a simulated CTL-wear model in rabbits/mice with eyelid closure that will allow experimental modulation of EH and PA binding without inflammation, injury or use of antimitotic agents. We will also use 3 strains of genetically altered mice (WT controls), which exhibit Bcl-2 overexpression (transgenic gain of function), Bcl-2 KO loss of Bcl-2 function) and a Bax KO mouse to establish a regulatory role for Bcl-2 protein(s) in EH and CTL-induced PA binding. Using our newly developed technologies (in vivo confocal; laser confocal triple fluorescence imaging) to assess EH in vivo and ex vivo, we will pursue 5 specific aims: (1) determine whether daily or de novo continuous CTL-wear produces least risk for MK or disturbance of EH in man; (2) establish a significant correlation between Bcl-2 expression and location, EH and risk for MK (PA binding) in normal rabbit and mouse (open, prolonged closed eye); (3) establish the same correlates as in (2) for prolonged periods (1,2 weeks) in a contact lens exposed rabbit cornea using test CTLs of differing types [rigid, soft, silicone hydrogel (Sih)] and lens-O2 transmission; (4) determine the status of EH in mice with stable TG gain of Bcl-2 function, Bcl-2 KO (loss of function) or the pro-apoptotic Bax KO (WT controls) and correlate changes in EH and MK risk (PA binding) to Bcl-2 expression location; (5) modulate corneal EH and PA binding levels in the TG and KO mice corneas by eyelid closure with/without prolonged CTL exposure with variable-O2, mouse-specific, test CTLs (1, 3, 7, 14 days), determine effects on Bcl-2 expression, and correlate changes in PA binding to EH and Bcl-2 expression/location.