去瓣型Epi—LASIK与LASEK术后角膜上皮内紧密连接蛋白occludin的表达

Expression of occludin in corneal epithelium after flap-free epipolis laser in-situ keratomileusis and laser- assisted subepithelial keratectomy

背景紧密连接作为角膜上皮屏障的重要构成部分，是角膜屈光手术后角膜上皮损伤修复中的关键因素，目前国内外有关这一方面的研究尚不多见。目的探讨和比较去瓣型微型角膜刀法准分子激光角膜上皮瓣下磨镶术（Epi—LASIK）与准分子激光上皮瓣下角膜磨镶术（LASEK）术后早期角膜上皮内紧密连接蛋白occludin的表达。方法48只健康清洁级成年新西兰白兔按照随机数字表法随机分为2个组，每组24只。2个组均取右眼分别行去瓣型Epi-LASIK和LASEK手术，后者术中也不保留角膜上皮瓣。于术后1、2、3、5d用空气栓塞法处死实验兔并分离角膜组织，每个时间点获取6只兔眼角膜标本。另取2只正常兔为正常对照组。采用免疫荧光检查法观察各组兔眼角膜上皮内occludin蛋白的表达变化，用RT．PCR半定量检测兔眼中央区角膜内occludinmRNA表达的变化。结果角膜免疫荧光检测结果表明，正常兔眼角膜上皮内可见occludin蛋白表达呈绿色荧光，荧光信号较强且排列有序。术后1～2d，LASEK组兔眼角膜上皮内绿色荧光信号表达强度低，数量少且排列紊乱，而去瓣型Epi—LASIK组角膜上皮内荧光信号强于LASEK组且排列整齐，术后3～5d，2个组荧光信号均增强，排列有序。RT—PCR检测表明，术后1、2、3d，LASEK组兔眼角膜上皮内occludinmRNA表达相对值分别为0．11±0．02、0．25±0．03、0．43±0．04，明显低于去瓣型Epi—LASIK组的0．20±0．04、0．44±0．04、0．76±0．04，差异均有统计学意义（t=6．476、12．898、17．315，P〈0．05）；术后5d，2个组角膜上皮内occludinmRNA表达相对值差异无统计学意义（t=-0．733，P〉0．05）。2个组随着术后时间的延长，occludinmRNA表达相对值均逐渐升高，总体差异有统计学意义（F时间=768．903，P=0．000）。结论与LASEK比较，去瓣型Epi—LASIK对角膜上皮紧密连接的损害轻，术后角膜上皮紧密连接的修复也更加迅速，提示去瓣型Epi—LASIK有利于减少术后角膜刺激症状和并发症的发生。

Background Tight junctions are thought to play a significant role in the maintenance of the corneal epithelial defense for the eye, and the restoration of the tight junctions is critical during epithelial wound healing after refractive surgery. However,there are few reports ahout this study. Objective The aim of this study was to investigate and compare the expression of occludin in corneal epithelium following flap-free epipolis laser in- situ keratomileusis （ Epi-LASIK ） and laser-assisted subepithelial keratectomy （ LASEK ）. Methods Forty-eight clean New Zealand white rabbits were randomized into 2 groups,and 24 rabbits for each group. Flap-free Epi-LASIK and LASEK were performed in the right eyes of the rabbits in two groups,and other 2 age matched normal rabbits were as normal controls. The animals were sacrificed and the corneal samples were obtained at 1,2,3,5 days after surgery. Immunofluoreseence staining was used to detect the expression of occludin in the corneal epithelium,and RT-PCR was used to identify the level of occludin mRNA in the central cornea. Results Occludin protein was expressed in normal corneal epithelium and showed the green fluorescence with the regular arrangement. The fluorescence intensity was lower in 1-2 days in LASEK group with the irregular arrangement; while the fluorescence signal in corneal epithelium was stronger in flap-free Epi-LASIK group. 3-5 days after surgery, the fluorescence intensities were both enhanced in two groups. RT-PCR showed that the relative expression of occludin mRNA in corneal epithelium was 0. 11±0.02,0.25 ±0.03,0.43±0.04 in LASEK group 1,2,3 days after surgery, respectively, and was lower than those of flap-free Epi-LASIK group （ 0.20 ± 0.04,0.44± 0.04,0.76 ±0.04 ） , showing significant differences between these two groups （ t = 6. 476, 12. 898, 17. 315, P〈O. 05 ）. No significant difference was seen in the expression of occludin mRNA in 5 days after surgery between two groups （t=-0. 733, P〉0.05 ）. The relative values of occludin mRNA expression in corneal epithelium were gradually increased with time prolongation, presenting a significant difference among various time points （ Ftime = 768. 903, P = 0. 000）. Conclusions The reformation of occludin in flap-free Epi-LASIK group is faster than that in LASEK group. Therefore, flap-free Epi-LASIK is prominant in reducing the stimulated symptoms and complication after the surgery.