【摘要】目的：研究基质金属蛋白酶抑制剂GM6001对大鼠角膜碱性烧伤后角膜新生血管的抑制作用。
方法：取健康成年Wistar大鼠20只，随机分成实验组和对照组，每组10只。两组均建立大鼠角膜碱性烧伤模型。实验组滴用GM6001滴眼液、氧氟沙星滴眼液和硫酸软骨素滴眼液；对照组滴用氧氟沙星滴眼液和硫酸软骨素滴眼液；各种滴眼液每日点眼4次，共7d。裂隙灯下观察角膜溃疡和角膜新生血管生长情况。
结果：碱烧伤后实验组和对照组出现新生血管的时间无显著差异（P>0.05）。新生血管的生长长度与生长面积，在伤后1d和3d，实验组和对照组无显著差异（P>0.05）；在伤后5d和7d，实验组和对照组有显著差异（P<0.05）。
结论：基质金属蛋白酶抑制剂GM6001点眼对角膜新生血管的生长有抑制作用。
【关键词】 基质金属蛋白酶抑制GM6001；角膜；新生血管
Restrained effect of GM6001 the tissue inhibitor of matrix metalloproteinases on the growth of rats corneal neovascularization after alkaline burn
QingCheng Shi, JianHua Ning
Shenyang Aier Hospital of Ophthalmology and Optometry, Shenyang 110003, Liaoning Province, China
Correspondence to:QingCheng Shi. Shenyang Aier Hospital of Ophthalmology and Optometry, Shenyang 110003, Liaoning Province, China. [email protected]
Abstract AIM: To study the restrained effect of GM6001 the tissue inhibitor of matrix metalloproteinases (TIMP) on the growth of rats corneal neovascularization after alkalineburn.
METHODS: Twenty Wistar rats were randomly divided into 2 groups, experimental group and control group, 10 rats in each group. In two groups, central corneas of rat eyes were burned by 1mol/L NaOH for 120 seconds. GM6001 eyedrops, Ofloxacin eyedrops and chondroitin sulfate eyedrops were used in experimental group. Ofloxacin eyedrops and chondroitin sulfate eyedrops were used in control group. Eyedrops were applied four times a day for one week in each group. The occurrence and development of CNV were observed by slitlamp microscope everyday. The length and the area of CNV were calculated.
RESULTS: The occurrence of CNV in experimental group was (1.40±0.05)d , and was (1.25±0.10)d in control, with no significant difference in the occurrence time of CNV（P>0.05）, in the length（P>0.05）and area of CNV at first day and third day after being burned. Significant difference（P<0.05）was found at fifth day and seventh day after being burned.
CONCLUSION: GM6001 restrains the progress of corneal ulcer and the growth of corneal neovascularization.
KEYWORDS: GM6001 the tissue inhibitor of matrix metalloproteinases; cornea; neovascularization
0引言
基质金属蛋白酶（MMP）是一组与角膜基质溶解密切相关的蛋白酶，GM6001是一种新型的人工合成的MMP抑制剂，对MMPS的抑制力远远强于其他常规药物如乙酰半胱氨酸、枸橼酸钠等。我们应用GM6001治疗大鼠角膜碱烧伤，探讨GM6001对碱烧伤后角膜溶解及角膜新生血管抑制作用的疗效。
1材料和方法
1.1材料
健康成年Wistar大鼠20只，无任何眼疾，体质量0.20～0.25kg，雌雄各半，购自佳木斯医学院。MMP抑制剂GM6001粉剂，二共药公司，日本东京，20g/L DMSO粉剂(二甲亚砜)，HEPES溶液(N2羟乙基哌嗪N′2乙磺酸)，佳木斯医学院药理实验室提供。裂隙灯显微镜，透射电子显微镜，佳木斯医学院提供。将GM6001粉剂置于含20g/L DMSO粉剂的HEPES缓冲溶液中，制成质量浓度为400mg/L的GM6001滴眼液（pH7.4），置于4℃冰箱中备用[1]。
1.2方法
将大鼠置于乙醚罐中1min后，称体质量，采用氯丙嗪12.5mg/kg，ip麻醉，固定大鼠，2.5g/L地卡因溶液点眼局部麻醉，将浸泡有1mol/L氢氧化钠溶液的直径为3.0mm圆形单层滤纸片，置于大鼠角膜中央2min，取下，用生理盐水冲洗结膜囊1min。将20只大鼠随机分成2组，每组10只，分别为实验组和对照组。实验组术后点氧氟沙星滴眼液和硫酸软骨素滴眼液及400mg/L的GM6001滴眼液；对照组术后点氧氟沙星滴眼液和硫酸软骨素滴眼液；每种滴眼液每日早、中、晚及夜间点眼4次，每种滴眼液时间间隔为20min。大鼠均于碱性烧伤后开始每日行裂隙灯显微镜下检查，分别记录于碱性烧伤后的1，3，5，7d角膜缺损面积，缺损深度以及角膜新生血管的生长情况。
1.2.1角膜新生血管定量分析
裂隙灯显微镜下检查，分别记录于碱性烧伤后的1，3，5，7d各组大鼠角膜新生血管长度和数量。测量时，以连续弯曲小、角膜新生血管朝向角膜混浊中心生长的最长血管为准，并计算角膜新生血管的生长面积A＝C/12×3.1416[r2(rl)2][2]，C为角膜新生血管累及角膜的圆周钟点数，l为角膜新生血管从角膜缘深入角膜的长度，r（大鼠角膜半径）＝3.5mm[3]。
1.2.2电子显微镜检查
取带有1mm宽巩膜的角膜组织用于透射电子显微镜的观察，标本切成1mm×6mm组织条块，置于20g/L戊二醛溶液固定2h后泡入二甲砷酸钠缓冲溶液中浸洗1d，然后以10g/L锇酸1h，再用二甲砷酸钠缓冲溶液冲洗，丙酮梯度脱水，环氧树脂618定向包埋，切片机超薄切片，电镜下观察大鼠角膜碱性烧伤后，再生上皮的厚薄、细胞排列极向及细胞间距。细胞间桥粒结构及细胞器（包括线粒体、粗面内质网和游离的核糖体、微管、微丝等）的改变。
统计学处理：数据使用SPSS10.0软件进行统计学检验并判断结果。
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