兔LASEK与PRK术后角膜Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的Western Blot研究

目的:用WesternBlot法比较准分子激光上皮下角膜磨削术(LASEK)与准分子激光角膜切削术(PRK)术后角膜Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的动态变化情况。方法:52只新西兰白兔分为8组,对每只兔右眼行LASEK,左眼行PRK。术后1d、7d、1个月、3个月、4个月、5个月、6个月观察Haze情况,处死动物取角膜行Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的动态变化过程的免疫组织化学检查与WesternBlot检测。结果:经免疫组化和WesternBlot研究发现:LASEK组术后角膜基质中Ⅰ、Ⅲ型胶原开始增生的时间早于PRK组,表达的强度与PRK组有显著差异;两组的Ⅴ、Ⅵ型胶原动态变化曲线相似,达到表达高峰的时间一致,PRK组Ⅴ、Ⅵ型胶原的表达均明显高于LASEK组,术后6个月时LASEK组表达显著弱于PRK组。结论:PRK手术后角膜基质中Ⅰ、Ⅲ、Ⅴ、Ⅵ型胶原的表达强度、达到高峰及恢复正常的时间与LASEK相比存在显著差异,表明PRK术后基质内有胶原的过量沉积,可能是临床PRK术后Haze严重及屈光回退的组织学基础。WesternBlot法是一种可比较准确地半定量检测胶原含量动态变化的研究方法。

Western Blot Analysis of Type Ⅰ、Ⅲ、Ⅴ、Ⅵ Collagen after Laser Epithelial Keratomileusis and Photorefractive Keratectomy in Cornea of Rabbits

PURPOSE: Use immunohistochemical staining and Western Blot analysis to observe and compare the accurate dynamic changes of type I, III, V, VI collagen in the wound healing processes of the rabbit cornea which underwent LASEK or PRK to investigate the possible mechanism of corneal haze and myopic regression. METHODS: New Zealand White rabbits were divided into 8 groups: normal control group (n=6), 1 day, 7 days, 1, 3, 4, 5 and 6 month groups (n=14). Every rabbit underwent LASEK in one eye while the other one with PRK. We use immunohistochemical staining and Western Blot analysis to compare the wound healing process of dynamic change of the type I, III, V and VI collagen in rabbit cornea of every time point. The results were analysised with data analysis software. RESULT: Immunohistochemical staining and Western Blot analysis showed that after LASEK, the cornea wound healing with type I and III collagen were much faster than PRK, and the wound response was also much weaker. Whereas for type V and VI collagen, their dynamic changes were resemble between LASEK and PRK, they both reached the peak value after 3 months since the surgery, but LASEK group returned to normal earlier than PRK. The value of these two types of collagen after PRK were higher than LASEK. The changes of these four types of collagen may offer us at least partial explaination to the difference between formation between corneal haze and refractive regression. CONCLUSIONS: There were significant differences between LASEK and PRK on type I, III, V and VI collagens or the time of reacting, reaching apex and returning to normal . LASEK had slighter intensity of reaction. The results indicate that there is excessive aggradation of collagens after PRK, it may be the histological foundation of obvious haze and myopia regression.