KERATOCYTE CRYSTALLIN PROTEINS AND CORNEAL TRANSPARENCY

赠款的长期目标是了解 角膜基质细胞晶体蛋白调节角膜透明度. 细胞水平。最近的研究表明，兔角膜基质细胞 表达转酮醇酶（transketolase，TKT）和醛（aldehyde）两种水溶性蛋白 脱氢酶1（ALDH 1）。此外，在初步研究中， 兔角膜创伤愈合和新生儿发育中， 这两种蛋白质是高度相关的反向散射 来自完整的活角膜中的角膜细胞的光。根据这些数据，PI 提出兔角膜基质细胞中的TKT和ALDH 1属于一类新的 角膜细胞特有的晶体蛋白质，其具有类似的结构， 调节角膜透明度的功能，如相关的晶体蛋白 在透镜中。总之，PI提出角膜细胞晶体蛋白的表达 蛋白质可以在细胞水平上控制角膜透明度，因此， 在疾病后角膜混浊和混浊的发展中起作用 和损伤，特别是准分子激光屈光性角膜切除术后， PI将通过以下实验目标来检验假设：（1） 鉴定和表征来自人假定的角膜细胞晶体蛋白， 牛、猪、小鼠和禽类角膜（SDS-Page法），胰蛋白酶肽序列 分析和蛋白质印迹以验证 角膜细胞晶体蛋白;（2）建立发育表达 角膜细胞晶体蛋白，并确定相对贡献 晶体蛋白表达对新生儿角膜发育的影响 与组织水合作用相比，眼睑打开后的透明度，角膜细胞 细胞密度、大小、厚度和分化，以及胶原纤维 （3）确定角膜基质细胞的时间表达 晶体蛋白在角膜冷冻损伤修复过程中的作用， 角膜细胞晶体蛋白的表达对来自角膜基质的光的反向散射的影响 完整活体角膜中角膜基质细胞;（4）建立角膜基质细胞表达 晶体蛋白，并确定光/暗和细胞因子的影响 刺激（TGF β、IL 1、FGF 2、PDGF）晶体蛋白表达;和 (5)局部应用调节角膜基质细胞晶体蛋白表达 细胞因子或抗细胞因子阻断抗体的应用 上调或阻断晶状体蛋白表达并确定对光的影响 角膜散光和角膜混浊。

The long-range goal of the grant is to understand the role of corneal keratocyte crystallin proteins in modulation of corneal transparency at the cellular level. Recent studies have shown that rabbit corneal keratocytes abundantly express two water soluble proteins, transketolase (TKT) and aldehyde dehydrogenase class 1 (ALDH1) in vivo. Furthermore, in preliminary studies of corneal wound healing and neonatal development in the rabbit, expression of these two proteins is highly correlated inversely to the backscattering of light from keratocytes in the intact, living cornea. Based on these data the PI proposes that TKT and ALDH1 in the rabbit keratocyte belong to a new class of crystalline proteins unique to the keratocyte, which serve a similar structural function in modulating corneal transparency, as do related crystalline proteins in the lens. Overall, the PI proposes that expression of keratocyte crystallin proteins may control corneal transparency at a cellular level, and therefore, play a role in the development of corneal clouding and haze following disease and injury, particularly after excimer laser photorefractive keratectomy, PRK. The PI will test the hypothesis through the following experimental aims: (1) identify and characterize putative keratocyte crystalline proteins from human, bovine, porcine, murine and avian corneas by SDS-Page, tryptic peptide sequence analysis and western blotting to verify the taxon specific expression of keratocyte crystalline proteins; (2) establish the developmental expression of keratocyte crystalline proteins and determine the relative contribution of crystalline protein expression to the neonatal development of corneal transparency following eyelid opening compared to tissue hydration, keratocyte cell density, size, thickness, and differentiation, and collagen fibril diameter and spacing; (3) determine the temporal expression of keratocyte crystalline proteins during repair of corneal freeze injury and correlate the expression of keratocyte crystallins to backscattering of light from keratocytes in intact, living cornea; (4) establish expression of keratocyte crystallins in vitro and determine the effects of light/dark and cytokine stimulation (TGFbeta, IL1, FGF2, PDGF) on crystalline protein expression; and (5) modulate keratocyte crystalline protein expression in vivo by topical application of cytokines or anti-cytokine blocking antibodies shown to upregulate or block crystallin expression and determine the effect on light scattering and corneal haze following PRK.