机械法准分子激光上皮瓣下角膜磨镶术与去瓣Epi-LASIK术后兔角膜基质细胞凋亡的研究

目的观察机械法准分子激光上皮瓣下角膜磨镶术（Epi-LASIK）与去瓣Epi-LASIK术后不同时间点角膜基质细胞的凋亡,探讨2种手术方式对角膜创伤修复过程的影响。方法 42只新西兰大白兔随机编号,其中40只兔各取一侧眼行Epi-LASIK,对侧眼行去瓣Epi-LASIK,剩余2只兔4只眼作为正常对照组。分别于术后4h,1、3、7d共4个时间点取出角膜组织,透射电镜下观察各组兔角膜组织的超微结构改变,采用TUNEL法检测2组兔手术后不同时间点角膜基质细胞的凋亡情况,应用免疫组织化学法检测2组兔手术后不同时间点白细胞介素-1β（IL-1β）在角膜基质细胞中的表达。结果 Epi-LASIK组术后1d及3d角膜基质细胞凋亡细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.42,P〈0.05;t=4.04,P〈0.05）;Epi-LASI组术后1、3、7d角膜基质中IL-1β表达的阳性细胞数明显高于去瓣Epi-LASIK组,差异均有统计学意义（t=2.13,P〈0.05;t=3.71,P〈0.05;t=3.06,P〈0.05）。Epi-LASIK组术后透射电镜下可见兔角膜基质细胞核固缩,染色质边集并可见凋亡小体。免疫组织化学染色显示Epi-LASIK组术后角膜基质中IL-1β的表达强于去瓣Epi-LASIK组。结论去瓣Epi-LASIK对角膜基质细胞的损害较Epi-LASIK轻,提示去瓣Epi-LASIK术后早期角膜创伤愈合反应的程度较轻,有利于角膜组织的快速修复。

Experimental study on keratocyte apoptosis following Epi-LASIK and off-flap Epi-LASIK in rabbits

Background Epipolis laser in situ keratomileusis（Epi-LASIK）is a new surface ablation procedure that is proved to be a safe,efficient and predictable for the treatment of myopia.Epi-LASIK is a major advancement over PRK and LASEK.Some surgeons thought that the epithelial flap should be removed after excimer laser ablation to reduce the development of corneal haze and postoperative pain.The theoretical advantages of ＇flap on＇ and ＇flap off＇ techniques of Epi-LASIK have not yet been studied.ObjectiveThis study was to investigate the effects of Epi-LASIK and off-flap epipolis laser in situ keratomileusis（off-flap Epi-LASIK）on the keratocyte apoptosis.MethodsEpi-LASIK was performed in the lateral eyes and off-flap Epi-LASIK was performed in the fellow eyes in 40 New Zealand white rabbits,and 4 normal eyes of 2 rabbits were used as controls.The corneal specimen was prepared at 4 hours and 1 day,3,7 days after operation.Morphological change of the cornea was observed by transmission electron microscope.Apoptosis of cornea stromal cell was detected by terminal deoxyribonucleotidyl transferase-mediated deoxynuridine triphosphate nick end labeling（TUNEL）assay,and the expression of interleukin-1β（IL-1β）in the corneal stroma was assessed by immunocytochemistry.ResultsThe number of TUNEL-positive cells was higher in Epi-LASIK group than that in off-flap Epi-LASIK group,showing statistically significant differences between them（t=2.42,P0.05;t=4.04,P0.05）.The expression level of IL-1β was enhanced in Epi-LASIK group in comparison with off-flap Epi-LASIK group at 1 day,3 and 7 days after surgery,and the differences presented the statistical significances（t=2.13,P0.05;t=3.71,P0.05;t=3.06,P0.05）.Karyopyknosis,chromatin margination and apoptosis bodies were seen in corneal stroma after Epi-LASIK under the transmission microscope.Immunochemistry revealed that IL-1β expression in corneal stroma was stronger after Epi-LASIK in comparison with off-flap Epi-LASIK.ConclusionOff-flap Epi-LASIK has less damage to corneal stomal cells in comparison with Epi-LASIK,and therefore the repair process of corneal wound is more quickly.