Gasymov Oktay K, Abduragimov Adil R, Prasher Pawan, Yusifov Taleh N, Glasgow Ben J
Department of Pathology and Ophthalmology, UCLA School of Medicine, Los Angeles, California, USA.
Invest Ophthalmol Vis Sci. 2005 Oct;46(10):3589-96. doi: 10.1167/iovs.05-0569.
Lipid contamination of the cornea may create an unwettable surface and result in desiccation of the corneal epithelium. Tear lipocalin (TL), also known as lipocalin-1, is the principal lipid-binding protein in tears. TL has been shown to scavenge lipids from hydrophobic surfaces. The hypothesis that TL can remove contaminating fatty acids and phospholipids from the human corneal surface was tested.
TL was purified from pooled human tear samples by size exclusion and ion exchange chromatographies. Tears depleted of TL were reconstituted from fractions eluted by size exclusion chromatography that did not contain TL. Fresh and formalin-fixed human corneas were obtained from exenteration specimens. Fluorescent analogs of either palmitic acid or phosphatidylcholine were applied to the corneal epithelial surface. Tears, TL, or tears depleted of TL were applied over the corneas, and spectrofluorometry and fluorescent stereomicroscopy were used to monitor the removal of fluorescent lipids. Tears used in the experiments were then fractionated by size exclusion chromatography to determine the component of tears associated with fluorescent lipids.
Significant enhancement of fluorescence for 16AP and NBD C(6)-HPC was evident in solutions incubated with whole tears and purified TL but not with tears depleted of TL for fixed and unfixed corneas. After the experiment, size exclusion fractions of tears showed that the fluorescence component coeluted with TL.
TL scavenges lipids from the human corneal surface and delivers them into the aqueous phase of tears. TL may have an important role in removing lipids from the corneal surface to maintain the wettability and integrity of the ocular surface.
角膜的脂质污染可能会形成不可湿润的表面，并导致角膜上皮干燥。泪液视黄醇结合蛋白（TL），也称为视黄醇结合蛋白-1，是泪液中主要的脂质结合蛋白。已证明TL可从疏水表面清除脂质。本研究对TL能否从人角膜表面清除污染的脂肪酸和磷脂这一假说进行了验证。
通过尺寸排阻色谱法和离子交换色谱法从汇集的人泪液样本中纯化TL。不含TL的泪液通过尺寸排阻色谱法洗脱的不含TL的级分进行重构。新鲜的和福尔马林固定的人角膜取自眼球摘除标本。将棕榈酸或磷脂酰胆碱的荧光类似物应用于角膜上皮表面。将泪液、TL或不含TL的泪液滴加在角膜上，采用荧光分光光度法和荧光立体显微镜监测荧光脂质的清除情况。然后通过尺寸排阻色谱法对实验中使用的泪液进行分级分离，以确定与荧光脂质相关的泪液成分。
对于固定和未固定的角膜，在与全泪液和纯化的TL孵育的溶液中，16AP和NBD C(6)-HPC的荧光有显著增强，但与不含TL的泪液孵育时则无此现象。实验后，泪液的尺寸排阻级分显示荧光成分与TL共洗脱。
TL可从人角膜表面清除脂质，并将其输送到泪液的水相中。TL在清除角膜表面脂质以维持眼表的湿润性和完整性方面可能具有重要作用。
