KERATOCYTE CRYSTALLIN PROTEINS AND CORNEAL TRANSPARENCY

赠款的长期目标是理解 角膜基质细胞晶状体蛋白在调节角膜透明度中的作用 在细胞水平上。最近的研究表明，兔角膜基质细胞 富含转酮醇酶(TKT)和乙醛两种水溶性蛋白 体内的脱氢酶1类(ALDH1)。此外，在对 兔角膜伤口愈合和新生血管发育的实验研究 这两种蛋白质的背向散射高度负相关。 来自完好无损的活体角膜的角膜基质细胞发出的光。根据这些数据，PI 提出兔角膜基质细胞中的TKT和ALDH1属于一类新的 角质形成细胞所特有的结晶蛋白，它们的结构相似 调节角膜透明度的功能，以及相关的晶体蛋白 在镜头里。总体而言，PI提出了角质细胞结晶蛋白的表达 蛋白质可能在细胞水平上控制角膜的透明度，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削术，PRK。 PI将通过以下实验目标来检验该假设：(1) 鉴定和鉴定人类角质形成细胞晶体蛋白， 牛、猪、小鼠和禽角膜的胰蛋白酶多肽序列分析 分析和Western blotting验证分类群的特异性表达 角质形成细胞晶体蛋白；(2)建立其在发育中的表达 并测定角质形成细胞晶体蛋白的相对贡献 晶体蛋白表达与新生儿角膜发育的关系 眼睑开放后透明度与组织水合、角质形成细胞的比较 细胞密度、大小、厚度、分化和胶原纤维 直径和间距；(3)决定角质形成细胞的时间表达 晶体蛋白在角膜冷冻损伤修复中的作用及相互关系 角质形成细胞晶状体蛋白对光的后向散射的表达 完整活体角膜上皮细胞；(4)建立角膜基质细胞的表达 晶体蛋白的体外培养及光/暗和细胞因子的影响 刺激(TGFbeta、IL1、FGF2、PDGF)对晶体蛋白表达的影响 (5)外用药物对体内角膜基质细胞晶体蛋白表达的影响 应用细胞因子或抗细胞因子阻断抗体显示 上调或阻断晶状体蛋白的表达并确定其对光的影响 准分子激光原位角膜磨镶术后散光和角膜混浊。

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.