三种干预措施下兔角膜基质中核黄素含量测定

Determination of riboflavin in rabbit corneal stroma under three interventions

背景EDTA是常用的眼用制剂添加成分，是一种重要的钙离子螯合剂，其对药物的角膜促渗作用可能为实现跨角膜上皮胶原交联提供便利，目前国内外关于EDTA对核黄素在角膜促渗方面的作用研究较少。目的比较去上皮、不去上皮以及使用促渗剂EDTA对核黄素在角膜基质渗透方面的影响，探讨EDTA在跨角膜上皮胶原交联中的可行性。方法清洁级健康无眼疾新西兰大白兔15只30只眼，应用随机数字表法将实验兔分为去上皮组、EDTA组（不去上皮，点质量分数0．5％EDTA—Na2 1h）和不去上皮组，每组5只实验兔。给予相应处理后，各组兔眼分别点质量分数0．1％核黄素＋质量分数20％右旋糖酐作用30min，随后去除EDTA组、不去上皮组角膜上皮，钻取各分组兔眼直径8．5mm的角膜中央组织，制备角膜组织匀浆上清液，采用高效液相色谱分析（HPLC）法对各组组织匀浆上清液进行核黄素定量检测。结果0．1％核黄素＋20％右旋糖酐点眼30min后，去上皮组、EDTA组、不去上皮组角膜基质中核黄素含量分别为（23．54±1．61）、（2．04±0．25）、（1．44±0．06）μg／g组织，不同方式处理后角膜组织中核黄素含量总体差异有统计学意义（F=1792．839，P=0．000），各组间两两比较差异均有统计学意义（t=41．780、7．484、43．408，P〈0．05）。除去上皮组角膜基质内核黄素含量超过理论值15μg／g组织以外，其余两组均未达到此理论值。结论交联术前去角膜上皮能充分提高核黄素在角膜基质内的弥散，促渗剂EDTA虽能有效促进核黄素在角膜的渗透，但尚不能取代去角膜上皮对促进核黄素角膜渗透方面的作用。

Backgroud EDTA is an important calcium chelator,commonly used as an ophthalmic additive, its role in promoting corneal penetration of drug may facilitate transepithelial corneal collagen crosslinking. However, there were few reports about the penetration of riboflavin to corneal stroma enhanced by EDTA both home and abroad. Objective To determine the concentrations of riboflavin in corneal intrastromal under different interventions using high-performance liquid chromatography （HPLC）. To ensure the efficacy and safety of 0.5% EDTA in riboflavin penetrating to corneal stroma by the transepithelial procedures. Methods Fifteen New Zealand white rabbits were randomized into three groups, corneal epithelium of 5 rabbits were debrided, corneal epithelium of another 5 rabbits were left in situ and 0.5% EDTA-Na2 was topically applied for 1 hour,and the corneal epithelium of last 5 rabbits were just left in situ,after those procedures 0. 1% riboflavin ＋ 20% dextran was applied topically to the corneas for 30 minutes（ 1 drop every 3 minutes）. Then,the epithelium in the corneas of EDTA and epi-situ group were carefully removed before all the corneas were puncbed with an 8.5 mm diameter blade and homogenized subsequently. Uhimately,the riboflavin concentrations were determined by HPLC. Results After treated with riboflavin for 30 minutes,the mean concentration of riboflavin in the epi-removed group was （23.54±1.61 ）μg/g tissue, the EDTA group was （ 2.04±0. 25 ）μg/g tissue and the epi-situ group was （ 1.44±0.06 ）μg/g tissue, showing significant difference among them （F= 1792. 839, P = 0. 000）. There were statistically significant differences between any two groups（ t= 41. 780,7. 484,43. 408, all at P〈0.05 ）. However, the riboflavin concentration of other two groups were failed to meet the theoretical value 15μg/g tissue,excepted for epi-removed group. Conclusions The diffusion of riboflavin to the corneal stroma can be fully enhanced by removing the epithelium preoperatively,penetration enhancer EDTA can effectively promote the penetration of riboflavin to the cornea, but it still can not replace the role of removing epithelium on the penetration of riboflavin to the cornea.