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，隐形眼镜（CTL）佩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，基底细胞保留时间延长和增殖减少。这些影响的净总和是产生“减慢”（EH）或“停滞”的上皮层，其特征是角膜中央上皮厚度减少。同时，ctl相关的缺氧也会导致角膜表面P. aeruginosa （PA）受体的上调，从而增加了发生微生物角膜炎（MK）的风险。基于这些发现，两个重要且相关的问题仍然存在：(1)哪种CTL佩戴计划对MK的风险最低，对EH的干扰最小？(2)正常角膜EH的调节机制是什么？ CTL是如何干扰这些机制的？最近的研究结果表明，Bcl-2蛋白的表达和位置（细胞质、细胞核）可能是这些事件的关键联系调控位点。为了验证这一假设，我们将在兔子身上使用体内CTL模型，并在兔子/小鼠的眼皮闭合中使用模拟CTL磨损模型，这将允许在没有炎症、损伤或使用抗有丝分裂药物的情况下实验性地调节EH和PA的结合。我们还将使用3株转基因小鼠（WT对照），它们表现出Bcl-2过表达（转基因功能获得），Bcl-2 KO功能丧失)和Bax KO小鼠，以确定Bcl-2蛋白在EH和ctl诱导的PA结合中的调节作用。使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）来评估体内和体外EH，我们将追求5个具体目标：(1)确定每天或从头开始连续佩戴ctl是否对人体MK或EH干扰的风险最小；(2)建立正常家兔和小鼠（睁眼、长时间闭眼）Bcl-2表达、定位、EH与MK （PA结合）风险的显著相关性；(3)使用不同类型的试验ctl[刚性、软质、硅水凝胶（Sih）]和晶状体- o2透射，在接触镜暴露的兔角膜中建立与(2)中相同的长时间（1,2周）相关性；(4)测定Bcl-2功能稳定TG增加、Bcl-2 KO（功能丧失）或促凋亡Bax KO （WT对照）小鼠的EH状态，以及EH和MK风险（PA结合）的变化与Bcl-2表达位置的相关性；(5)通过闭合眼睑调节TG和KO小鼠角膜EH和PA结合水平，同时/不同时暴露于变氧、小鼠特异性CTL（1、3、7、14天），确定对Bcl-2表达的影响，以及PA结合EH和Bcl-2表达/位置的变化。

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.