Ophthalmology Unit, University of Bologna, Bologna, Italy.
Eye (Lond). 2010 Aug;24(8):1396-402. doi: 10.1038/eye.2010.7. Epub 2010 Feb 12.
To analyze tear protein variations in patients suffering from dry eye symptoms in the presence of tear film instability but without epithelial defects.
Five microlitres of non-stimulated tears from 60 patients, suffering from evaporative dry eye (EDE) with a break-up time (BUT) <10 s, and from 30 healthy subjects as control (no symptoms, BUT >10 s) were collected. Tear proteins were separated by mono and bi-dimensional SDS-PAGE electrophoresis and characterized by immunoblotting and enzymatic digestion. Digested peptides were analyzed by liquid chromatography coupled to electrospray ionization quadrupole-time of flight mass spectrometry followed by comparative data analysis into Swiss-Prot human protein database using Mascot. Statistical analysis were performed by applying a t-test for independent data and a Mann-Whitney test for unpaired data (P<0.05).
In EDE patients vscontrols, a significant decrease in levels of lactoferrin (data in %+/-SD): 20.15+/-2.64 vs 24.56+/-3.46 (P=0.001), lipocalin-1: 14.98+/-2.70 vs 17.73+/-2.96 (P=0.0001), and lipophilin A-C: 2.89+/-1.06 vs 3.63+/-1.37 (P=0.006) was revealed, while a significant increase was observed for serum albumin: 9.45+/-1.87 vs 3.46+/-1.87 (P=0.0001). No changes for lysozyme and zinc alpha-2 glycoprotein (P=0.07 and 0.7, respectively) were shown. Proteomic analysis showed a downregulation of lipophilin A and C and lipocalin-1 in patients, which is suggested to be associated with post-translational modifications.
Data show that tear protein changes anticipate the onset of more extensive clinical signs in early stage dry eye disease.
分析患有泪膜不稳定但无上皮缺损的干眼症患者的泪液蛋白变化。
收集 60 例患有蒸发性干眼症（EDE）的患者（BUT<10s）和 30 例健康对照者（无症状，BUT>10s）的非刺激性泪液 5μl。通过单维和二维 SDS-PAGE 电泳分离泪液蛋白，并通过免疫印迹和酶消化进行鉴定。通过液相色谱-电喷雾电离四极杆飞行时间质谱联用和 Mascot 对瑞士Prot 人蛋白数据库进行比较数据分析，对消化肽进行分析。采用独立数据 t 检验和非配对数据 Mann-Whitney 检验进行统计学分析（P<0.05）。
与对照组相比，EDE 患者的乳铁蛋白（%+/-SD）水平显著降低：20.15+/-2.64 vs 24.56+/-3.46（P=0.001），脂联素-1：14.98+/-2.70 vs 17.73+/-2.96（P=0.0001），脂磷蛋白 A-C：2.89+/-1.06 vs 3.63+/-1.37（P=0.006），而血清白蛋白水平显著升高：9.45+/-1.87 vs 3.46+/-1.87（P=0.0001）。溶菌酶和锌-α-2 糖蛋白无变化（P=0.07 和 0.7）。蛋白质组学分析显示，患者的脂磷蛋白 A 和 C 以及脂联素-1 表达下调，提示其与翻译后修饰有关。
数据表明，泪液蛋白变化预示着早期干眼症更广泛的临床症状的发生。
