兔Epi-LASIK上皮瓣弃留对角膜基质TGF-β_1、bFGF表达及细胞凋亡的影响

Effects of removal or reserve rabbit epithelial flap in Epi-LASIK on expression of TGF-β_1,bFGF and apoptosis of corneal stroma cells

目的比较兔机械法激光上皮下角膜磨镶术(epithelial laser in situ keratomileusis,Epi-LASIK)弃留上皮瓣对角膜基质转化生长因子-β1(transforming growth factorβ1,TGF-β1)、碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)表达水平及细胞凋亡的影响,进而探讨上皮瓣对角膜创伤愈合过程的影响。方法 21只新西兰大白兔行保留上皮瓣Epi-LASIK(留瓣组),21只行弃上皮瓣Epi-LASIK(弃瓣组)。分别于术后4 h、1 d、3 d、5 d、7 d、15 d、30 d每组各处死3只(6眼)兔,通过免疫组织化学和TUNEL法检测兔角膜基质中TGF-β1、bFGF的表达及基质细胞凋亡水平,并观察各组术后角膜上皮愈合时间和haze发生情况。结果弃瓣组上皮愈合时间为(3.88±0.98)d,留瓣组为(4.05±0.78)d,差异无统计学意义(t=0.640,P=0.526)。术后两组均未发现3级及以上haze产生;术后3 d,两组haze发生率差异有统计学意义(χ2=4.269,P=0.039),留瓣组较弃瓣组haze程度重;术后30 d,两组haze均消失。各时间点留瓣组TGF-β1、bFGF阳性细胞数均多于弃瓣组,但组间差异均无统计学意义(均为P>0.05)。术后4 h,留瓣组基质细胞凋亡细胞数(23.63±5.72)多于弃瓣组(16.75±4.07),差异有统计学意义(t=2.400,P=0.037);余时间点两组间差异均无统计学意义(均为P>0.05)。结论 Epi-LASIK保留上皮瓣增加基质细胞凋亡水平和术后haze发生率,上皮瓣是角膜创伤愈合修复过程的负性影响因素之一。

Objective To discuss the effects of removal or reserve the corneal epithelial flap in epithelial laser in situ keratomileusis(Epi-LASIK)on expression of transforming growth factor-β_1(TGF-β_1),basic fibroblast growth factor(bFGF)and apoptosis of corneal stroma cells and further investigate the effects of epithelial flap on corneal wound healing.Methods Twenty-one rabbits were operated by traditional reserve epithelial flap Epi-LASIK(reserve flap group),and 21 rabbits with removal epithelial flap Epi-LASIK(removal flap group).Each three rabbits(6 eyes)in each group were sacrificed at postoperative 4 hours,1 day,3 days,5 days,7 days,15 days and 30 days,immunohistochemistry and TUNEL methods were used to detect the expression of TGF-β_1,bFGF and apoptosis of corneal stroma cells,and corneal epithelial healing time and haze were observed postoperatively.Results The corneal epithelial healing time of removal flap group and reserve flap group were(4.05±0.78)days and(3.88±0.98)days,respectively there was no statistical difference(t=0.640,P =0.526).No haze with or more than grade three was found in two groups,there was statistical difference in rate of haze at postoperative 3 days between two groups(x^2=4. 269,P=0.039),all haze disappeared at postoperative 30 days.The TGF-β_1 and bFGF positive cells at postoperative each time points in reserve flap group were more than those in removal flap group,but there was no statistical difference between two groups(all P>0.05).The number of apoptotic cell at postoperative 4 hours in reserve flap group(23.63±5.72)was more than that in removal flap group(16.75±4.07),there was statistical difference(t=2. 400,p=0.037)and no difference was found at other time points(all P>0.05).Condusion Reserving corneal epithelial flap in Epi-LASIK will increase the stroma cell apoptosis and incidence rate of haze,the epithelial flap is one of the negative factors for corneal wound healing.