组织蛋白酶B抑制剂干预鼠视网膜新牛血管形成的研究

目的 探讨组织蛋白酶B(Cathepsin B)抑制剂CA-074Me对视网膜新生血管形成的干预作用.方法 实验研究.将C57BL/6J 7日龄小鼠60只用随机数字表法分为正常对照组、空白对照组、阴性对照组和实验组,每组15只,空白对照组、阴性对照组和实验组在缺氧环境中建立视网膜新生血管模型.实验组小鼠出氧箱后每眼球周注射CA-074Me 5 mg·kg-1·d-1,阴性对照组球周注射与实验组溶剂等量的二甲基亚砜,正常对照组、空白对照组均不给予任何药物,连续5 d.测定眼组织组织蛋白酶B活性;通过免疫组织化学的方法 观察组织蛋白酶B在视网膜组织的表达;通过HE染色法测定不同组突破视网膜内界膜的血管内皮细胞核数目;通过视网膜ADP酶染色铺片,测量无血管区面积、无血管区面积与视网膜面积的比值.结果 正常对照组、空白对照组、阴性对照组、实验组组织蛋白酶B活性分别为(17.75±2.30)、(28.75±3.14)、(29.16±2.78)、(23.14±3.53)nmol·min-1·mg-1,实验组分别与各对照组比较,两组之间差异均有统计学意义(F=13.16,P＜0.01),阴性对照组与空白对照组比较差异无统计学意义(P＞0.05);免疫组化测定正常对照组、空白对照组、阴性对照组和实验组组织蛋白酶B平均吸光度值分别为0.24±0.02、0.35±0.05、0.36±0.07、0.35±0.01,实验组、空白对照组、阴性对照组之间两两比较,差异均无统计学意义(P＞0.05);视网膜石蜡切片HE染色后,正常对照组、空白对照组、阴性对照组、实验组平均每张切片中突破内膜新生血管内皮细胞核数分别为2.71±1.07、67.51±11.55、65.16±5.78、27.14±3.53,实验组与各对照组比较差异均有统计学意义(F=9.12,P＜0.01),阴性对照组与空白对照组比较差异无统计学意义(P＞0.05);正常对照组、空白对照组、阴性对照组、实验组无血管区面积分别为(1.17±0.30)、(5.34±0.74)、(5.16±0.68)、(2.38±0.53)mm2,实验组与各对照组比较差异均有统计学意义(F=7.57,P＜0.01),阴性对照组与空白对照组比较差异无统计学意义(P＞0.05);正常对照组、空白对照组、阴性对照组和实验组无血管区面积/视网膜面积分别为0.09±0.01、0.24±0.03、0.23±0.07,0.16±0.05,实验组与各对照组比较差异均有统计学意义(F=8.32,P＜0.01),阴性对照组与空白对照组比较差异无统计学意义(P＞0.05).结论 组织蛋白酶B抑制剂CA-074 Me对C57BL/6J小鼠视网膜新生血管形成有一定程度的抑制作用,有可能成为治疗视网膜新生血管的一种新的方法 .(中华眼科杂志,2008,44:207-211)

The experimental study of specific inhibitor-CA-074Me of Cathepsin B suppressing retinal neovascularization

OBJECTIVE: To observe the effect CA-074Me on the retinal neovascularization of CS7BL/6J with retinal neovascularization, and to explore the new way of treatment for retinal neovascularization. METHODS: It was a experimental study. Sixty-seven-days-old C57BL/6J mice were divided into four groups randomly including normal control group, blank control group, negative control group, experiment group. Blank control group, negative control group, experiment group were exposed to 75% oxygen to established a model of retinal neovascularization. Experiment group were injected with CA-074Me (5 mg x kg(-1) x d(-1)) by periocular injection one time every eye for 5 days after mice left oxygen box. Negative control group were received the same dose of DMSO at the same time. Normal control group and blank control group did not received any medicine. The Cathepsin B activity of the tissue of mice'eye were measured. The mean optical density of Cathepsin B of retina were measured by immunohistochemistry; the number of new vascular cell nuclei extending into the internal limiting membrane in cross-sections was measured. Retinal neovascularization were tested by the vascular pattern in adenosine diphosphate-ase (ADPase) stained retina flat-mounts. RESULTS: CA-074Me reduced the Cathepsin B Activity, the number of new vascular cell nuclei extending into the internal limiting membrane, the non-vascular area of retina, and the ratio of the nonvascular area of retina and the whole retina area. CONCLUSION: The specific inhibitor-CA-074Me of Cathepsin B can reduce the retinal neovascularization of C57BL/6J to some extent, may be a new treatment for retinal neovascularization in the future.