微型角膜刀法准分子激光角膜上皮瓣下磨镶术后兔角膜上皮瓣结构及活性的研究

目的 探讨角膜微型刀上皮瓣下准分子激光原位角膜磨镶术（Epi-LASIK）术后上皮瓣形态结构、活性的变化、周边上皮（上皮瓣切口缘与角膜缘之间的区域）增生情况及其对细胞凋亡的影响。方法对29只新西兰白兔的双眼进行手术,28只眼行Epi—LASIK,24只眼行PRK,随机分为4组,在术后1、3、5、7d取标本,6只未手术眼作为空白对照组。采用透射电镜、光镜观察形态结构的变化;冰冻切片行酶组织化学检测上皮瓣细胞活性的变化;石蜡切片行凋亡及增殖细胞核抗原免疫组织化学检测。结果透射电镜发现KM5000D型上皮刀分离的上皮瓣基底膜完整,细胞之间结合紧密,术后上皮瓣与基质粘合牢固。1、3、5、7d上皮瓣细胞三磷酸腺苷酶和葡萄糖．6．磷酸酶活性（上皮瓣与周边上皮细胞酶活性比值）分别为79％、58％、69％、86％和79％、63％、77％、97％;各组Epi-LASIK眼周边上皮细胞活性与空白对照组差异无统计学意义（F=1．09,P〉0．05）。各组Epi-LASIK眼周边上皮增生与空白对照差异无统计学意义（F=1．10,P〉0．05）。Epi-LASIK和PRK组在术后1d基质细胞凋亡数为（3．429±1．693）和（3．796±1．998）个／10000μm^2,差异无统计学意义（t=-0．33,P〉0．05）;而Epi-LASIK眼术后3、5、7d基质细胞凋亡少于PRK,差异均有统计学意义（P〈0．01）。结论KM5000D型上皮刀分离的上皮瓣结构完整,细胞结合紧密,能保持较高活性,无明显刺激周边角膜上皮增生,有助于抑制凋亡的发生。（中华腠科杂志,2007,43：651-657）

The structure and viability analysis of corneal epithelial flap in the rabbit cornea after Epi-LASIK

OBJECTIVE: To evaluate the changes of ultrastructure and viability in the rabbit corneal epithelial flap after Epi-LASIK (epipolis laser in-situ keratomileusis) surgery and its effect on keratocyte apoptosis and proliferation of peripheral corneal epithelium (out of corneal epithelial flap). METHODS: Fifty-eighty eyes of 29 New Zealand rabbits were used, Epi-LASIK was performed in 28 eyes and Photorefractive keratectomy (PRK) was carried out in 24 eyes. treated-eyes were randomly divided into four groups and were sacrificed at 1, 3, 5, 7 days after surgery, six eyes without treatment were served as blank controls. Histological structure from The specimens of Epi-LASIK and controls eyes were assessed by light, transmission electron microscopy; epithelial cells viability were assessed by enzyme-histochemistry and Immunohistochemistry staining (proliferating cell nuclear antigen, PCNA) were performed to detect proliferation of peripheral corneal epithelial cells. Apoptotic cells were detected by TUNEL assay (TdT-mediated dUTP nick-end labeling) from the specimens of Epi-LASIK and PRK. RESULTS: The study from Transmission electron microscopy demonstrated that epithelial flap separated by KM5000D type epikeratome retained its typical stratification and integrity and the basement membrane including lamina densa and lamina lucida were compaginated with stroma. The expression of ATP enzyme, G-6-P enzyme from epithelial flap to peripheral epithelium of Epi-LASIK-treated eyes were (79%, 58%, 69%, 86%), (79%, 63%, 77%, 97%) at 1, 3, 5, 7 days after surgery respectively. There was no statistically significant difference in the cell Viability and the number of PCNA in peripheral epithelial cells among four Epi-LASIK groups and control group. At 1 day after surgery, no difference in TUNEL positive cells were seen between specimens of Epi-LASIK (3.429 +/- 1.693) and PRK (3.796 +/- 1.998); At 3, 5, 7 days, there was a significant difference in the number of keratocyte apoptosis in PRK compared to Epi-LASIK specimens. CONCLUSIONS: Epithelial flap separated by KM5000D type epikeratome retained its typical stratification and integrity. The flap keeps high viability and no peripheral epithelial cell proliferation and therefore it may play a role in the inhibition of keratocyte apoptosis.