来氟米特活性代谢物A771726抑制角膜新生血管的实验研究  

Inhibition of corneal neovascularization by the active metabolite of leflunomide A771726

作　　者：郝念[1] 张明昌[1] 

机构地区：[1]华中科技大学附属协和医院眼科,武汉430022

出　　处：《眼科研究》2008年第3期191-195,共5页Chinese Ophthalmic Research

摘　　要：目的探讨来氟米特活性代谢物丙二酸次氮酰胺(A771726)对角膜新生血管的抑制作用及机制。方法(1)碱烧伤法制作大鼠角膜新生血管模型,40只SD大鼠随机分成A、B、C及D组,每组10只大鼠(10只眼)。空白对照组(A组)生理盐水滴眼,B、C、D组伤后分别滴用0·5%、1·0%及2·0%的A771726滴眼液,均每日4次,共28d。每日裂隙灯显微镜下观察角膜新生血管的生长情况,测量新生血管面积。(2)不同浓度A771726与体外培养的人脐静脉内皮细胞(ECV-304)共同孵育,以四唑盐比色法(MTT法)检测细胞的增生活性,激光共聚焦显微镜检测免疫荧光染色增生细胞核抗原(PCNA)的表达。结果(1)C、D组大鼠角膜新生血管面积均显著小于空白对照A组(P<0·01),而B组大鼠角膜新生血管面积与A组差异无统计学意义(P>0·05)。(2)40、80、160、320μmol/LA771726与人脐静脉内皮细胞共同孵育36h可抑制细胞增生,随浓度增加抑制作用增强,免疫荧光染色显示细胞核PCNA的表达有降低。结论A771726能通过抑制血管内皮细胞增生阻止角膜新生血管生成。Objective This paper was to study the inhibitory effects of A771726,the active metabolite of leflunomide,on corneal neovascularization. Methods The corneal neovascularization was induced by corneal alkali burn with 1 mol/L NaOH solution in 40 SD rats. The rats were randomly divided into 4 groups and 10 rats for each. Group A was treated with 0. 9% sodium chloride as blank control, and 0. 5% , 1.0% and 2. 0% A771726 eye drops was topically administrated in group B,group C and group D 4 times per day from day 0 to 28 following alkali hum. The occurrence and development of corneal neovascularization were observed every day with slit lamp microscope. For the in vitro study, cultured human umbilical vein endothelial cells （ECV-304） were incubated with different concentrations of A771726 solution. The growth inhibition of cells was assessed by methyl thiazolyl tetrazolium （MTT） ,and the expression of proliferation cell nuclear antigen （PCNA） was detected using immunofluorescence under the laser confocal microscope. Results The corneal neovasculafization area in group C and D was significantly smaller than that in the group A in 4,7,14,21 and 28 days after corneal alkali burn （P 〈 0.01 ） , and no significant difference was found in corneal neovascularization area between group A and group B （P 〉 0.05 ）. The inhibiting rate of 0. 5% , 1.0% and 2.0% A771726 eye drops was 4. 15% ,59. 96% and 61.01% respectively. The OD value of human umbilical vein endothelial cells was gradually lowed in 40,80,160,320 μmol/L A771726 co-incubation for 36 hours groups in a concentration-dependent manner,presenting a significant difference in comparison with group A （P 〈 0.01 ）. The green fluorescence of PCNA in cuhured cells was weakened significantly as the increase of A771726 concentration. Conclusion A771726 inhibits the proliferation of vascular endothelial cells,which may be one of the mechanisms underlying inhibitory effect of A771726 on growth of corneal neovascularization.

关 键 词：丙二酸次氮酰胺 角膜新生血管 人脐静脉内皮细胞 激光共聚焦显微镜 

分 类 号：R772[医药卫生—眼科] R770.1[医药卫生—临床医学]