基质金属蛋白酶2及其抑制剂在大鼠青光眼滤过术后滤过泡组织中的动态表达

背景 青光眼滤过术后瘢痕形成是导致手术失败的主要原因,探讨球结膜滤过泡中基质金属蛋白酶2(MMP-2)及组织金属蛋白酶抑制剂2(TIMP-2)的表达对于术后瘢痕形成的发病机制研究具有重要意义. 目的 观察大鼠球结膜滤过泡模型滤过区组织中MMP-2及TIMP-2的表达与瘢痕形成进程的关系.方法 采用完全随机化分组法将63只6～8周龄清洁级雄性SD大鼠分为正常对照组和术后1、3、5、7、14、28 d组,每只大鼠任选一侧眼为实验眼.采用前房引流管植入术建立大鼠球结膜滤过泡模型,术后裂隙灯显微镜下观察大鼠球结膜滤过泡的形成情况及眼前节反应,各组于相应时间点颈椎脱臼法处死大鼠,其中3只眼制备完整眼球冰冻切片,采用免疫荧光化学染色法检测标本中MMP-2及TIMP-2的表达和定位;另取6只大鼠结膜滤过区组织行Western blot检测,观察MMP-2和TIMP-2在滤过区组织中的动态表达. 结果 所有术眼在术后第1天均形成不同程度隆起的滤过泡,维持时间为7 ～17d,平均生存时间为12d.Western blot检测结果显示,正常对照组大鼠滤过区组织中MMP-2蛋白的相对表达量为121.67±4.37,在术后3d组开始逐渐增高,为183.67±5.61,术后7d组达峰值,为230.50±8.48,随后逐渐降低,术后28 d组为172.33±8.43,仍高于正常对照组,各组大鼠滤过区组织中MMP-2蛋白的相对表达量总体比较差异有统计学意义(F=280.18,P＜0.05).正常对照组大鼠滤过区组织中TIMP-2蛋白的相对表达量为102.50±6.25,在术后3d组开始明显升高,为162.67±7.00,术后5d组TIMP-2蛋白表达量达峰值,为232.00±11.03,之后逐渐下降,术后28 d组降至150.50±6.41,但仍高于正常对照组,各组间滤过区组织中TIMP-2蛋白的相对表达量的总体比较差异有统计学意义(F=145.34,P＜0.05).免疫荧光组织化学结果显示,正常对照组MMP-2、TIMP-2仅在大鼠结膜上皮层微弱表达,呈红色荧光,而在术后大鼠滤过区结膜组织上皮层和固有层均呈强荧光. 结论 MMP-2和TIMP-2在大鼠青光眼滤过术后滤过区组织的动态表达与滤过泡的纤维化过程相一致,提示MMP-2和TIMP-2参与结膜滤过泡的瘢痕化过程.

Dynamic expressions of matrix metalloproteinase-2 and its inhibitor in conjunctival filtering blebs of rats following glaucoma filtering surgery

Background The scarring of conjunctival filtering blebs after glaucomatous surgery is a leading cause of operation failure.Exploring the balance between matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in conjunctival filtering bleb is very important for the study on pathogenesis of postoperative scarring.Objective This study was to evaluate the role of MMP-2 and TIMP-2 in wounding healing of subconjunctival tissue after filtering surgery in rats.Methods Sixty-three clean male SD rats were divided into normal control group and postoperative 1-day,3-day,5-day,7-day,14-day and 28-day group.The drainage tube was monocularly implanted into the anterior chamber of the rats to establish the filtering surgery models,and the operative response of the eyes was examined under the slit lamp microscope.The animals were sacrificed in corresponding time points,and the frozen sections of eyeballs were prepared,and the expressions of MMP-2 and TIMP-2 in conjunctival and subconjunctival tissues were detected using immunofluorescence technique.Western blot was employed to assay the dynamic expressions of MMP-2 and TIMP-2 proteins in conjunctival and subconjunctival tissues in different groups.The use and care of the rats complied with the Instruction Notions with Respect to Care for Laboratory by State Ministry of Science and Technology.Results Filtering blebs were formed in all the operated eyes 1 day after surgery and remained for 7-17 days,with the average survival time of 12 days.Western blot assay revealed that the expression levels of MMP-2 protein in the filtering blebs of the normal control group were 121.67 ±4.37,and the expressions were gradually elevated from the postoperative 3-day group (183.67±5.61) until the postoperative 7-day group (230.50±8.48) and then gradually declined till the postoperative 28-day group (172.33 ± 8.43),showing a significant difference among the groups (F=280.18,P＜0.05).In addition,the expression levels of TIMP-2 protein in filtering blebs of the normal control group were 102.50 ±6.25.The expression levels were gradually raised in postoperative 3-day group (162.67±7.00) and peaked in the postoperative 5-day group (232.00± 11.03),and then the levels gradually reduced till the postoperative 28-day group (150.50±6.41),with a significant difference among the groups (F =145.34,P ＜ 0.05).Immunofluorescent staining showed that MMP-2 and TIMP-2 were weakly expressed in the conjunctival epithelium of the normal rats,while in the operated rats,strong fluorescence for MMP-2 and TIMP-2 was seen in both conjunctival epithelium and subconjunctival tissues of filtering blebs.Conclusions The expression trends of MMP-2 and TIMP-2 in the filtering blebs are consistent to the fibrosis process of conjunctival tissue,indicating that MMP-2 and TIMP-2 participate in the scar formation of conjunctival filtering bleb after glaucoma filtering surgery.