塞来昔布与羊膜匀浆提取液对兔角膜热烧伤后角膜新生血管面积及基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)表达的影响

Effects of celecoxib and amniotic membrane suspension on neovascularization area and expression of MMP-2,mmp-9 in corneal thermal-burned rabbit

目的比较塞来昔布与羊膜匀浆提取液对兔角膜热烧伤后角膜新生血管(corneal neovascularization,CNV)面积及基质金属蛋白酶-2(matrix metallo-proteinase-2,MMP-2)、基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的影响,为临床上塞来昔布治疗CNV提供理论依据。方法建立36只兔角膜热烧伤动物模型,随机分为3组:阴性对照组、羊膜匀浆提取液组、塞来昔布组,每组12只,8只用于观察,4只用于取材。各组兔均于造模后第1天开始球结膜下注射,至造模后第14天;阴性对照组注入90 g·L^(-1)生理盐水0.1 mL,羊膜匀浆提取液组注入羊膜匀浆提取液0.1 mL,塞来昔布组注入8 mg·mL^(-1)塞来昔布溶液0.1 mL。对比热烧伤后4 d、7 d、14 d在裂隙灯显微镜下所观察到的各组角膜的组织形态及新生血管的生长状况,并测算CNV面积;各组均于热烧伤后第7天随机取4只兔完整角膜,行免疫组织化学染色并用计算机图像分析系统检测MMP-2、MMP-9的表达。结果角膜热烧伤后第4天,阴性对照组、羊膜匀浆提取液组、塞来昔布组都可见不同程度的角膜水肿,角膜缘血管充血扩张,新生的小血管芽呈毛刷状,垂直角膜缘切线向内生长。热烧伤后4 d、7 d、14 d,CNV面积阴性对照组为(11.32±1.11)mm^2、(38.49±4.64)mm^2、(43.30±4.39)mm^2;羊膜匀浆提取液组为(9.69±1.30)mm^2、(31.15±4.85)mm^2、(37.19±5.27)mm^2;塞来昔布组为(8.47±1.20)mm^2、(30.31±4.93)mm^2、(36.69±3.54)mm^2。热烧伤后4 d、7 d、14 d,阴性对照组的CNV面积均明显大于羊膜匀浆提取液组和塞来昔布组,差异均有统计学意义(均为P<0.05);羊膜匀浆提取液组的CNV面积与塞来昔布组相比,差异均无统计学意义(均为P>0.05)。热烧伤后第7天MMP-2和MMP-9表达阴性对照组大于羊膜匀浆提取液组和塞来昔布组,差异均有统计学意义(均为P<0.05);羊膜匀浆提取液组与塞来昔布组相比,差异均无统计学意义(均为P>0.05)。结论塞来昔布与羊膜匀浆提取液均能有效抑制角膜热烧伤后新生血管的生长,可能与其抑制MMP-2、MMP-9在热烧伤后角膜中的表达有关。

Objective To compare the effects of celecoxib and amniotic membrane suspension （AMS） on corneal neovascularization （CNV） area and expression of matrix metalloproteinase-2 （MMP-2） and matrix metalloprotein-ase-9 （MMP-9） in the growth of corneal neovascularization after thermal burn in rabbits,and provide a theoretical basis of celecoxib for the clinical treatment of corneal neovascularization.Methods Left corneas of 36 rabbits were burned by the home-made burning-device,and randomly divided to three groups：negative control group （n=12）,AMS group （n=12） and celecoxib group （n=12）,were respectively sub-conjunctival injected by 90 g·L-1 saline （0.1 mL）,AMS （0.1 mL） and 8 mg·mL-1 celecoxib solution （0.1 mL）.The histological morphology,growth condition and area of CNV were compared under slit lamp microscope at 4 days,7 days and 14 days after thermal-burned.At 7 days after thermal-burned,four appropriate corneas were randomly taken to detect the expression of MMP-2 and MMP-9 by immunohistochemistry,and the results were analyzed by computer image analysis system.Results At 4 days,7 days,14 days after thermal-burned,the areas of neovascularization were （11.32±1.11）mm2,（38.49±4.64）mm2,（43.30±4.39）mm2in negative control group,（9.69±1.30）mm2,（31.15±4.85） mm2,（37.19±5.27）mm2in AMS group,（8.47±1.20）mm2,（30.31±4.93）mm2,（36.69±3.54）mm2in celecoxib group,respectively.At different time points,neovascularization area in AMS group or celecoxib group was significantly lower than negative control group （all P〈0.05）.There was no difference between AMS and celecoxib group （all P〉 0.05）.At 7 days after thermal-burned,the expression of MMP-2 and MMP-9 was not different between AMS group and celecoxib group （all P〉0.05）,and significantly lower than negative control group （all P〈0.05）.Conclusion Celecoxib and amniotic membrane suspension can all effectively inhibit CNV after thermal-burned,which may be related to the down-regulated expression of MMP-2,MMP-9 in thermal-burned corneas.