利鲁唑对体外培养人脐静脉内皮细胞及小鼠视网膜新生血管形成的影响

Effects of riluzole on retinal neovascularization and in vitro cultured human umbilical vein endothelial cells

背景：研究已证实利鲁唑具有较强的抑制蛋白激酶C-βⅡ单体介导血管内皮细胞生长因子促内皮细胞增殖效应的作用，因此推测其可用于抑制视网膜新生血管的形成。目的：观察利鲁唑对培养的人脐静脉内皮细胞增殖及小鼠视网膜新生血管形成的抑制及其作用途径。设计、时间及地点：单一样本观察及随机对照动物实验，于2007—08／12在重庆医科大学动物实验室及基础研究实验室完成。材料：清洁级出生第3天的健康C57BL／6小鼠30只60眼，雌雄不限，以改良法建立高氧诱导血管增生性视网膜病变小鼠模型。利鲁唑原料物质为北京万全医药科技有限公司产品，批号：060630，含量≥98．5％。方法：常规传代培养人脐静脉内皮细胞，随机分为3组。高氧模型组及利鲁唑治疗组为造模小鼠，利鲁唑治疗组小鼠腹腔注射利鲁唑10mg／（kg·d），正常对照组小鼠腹腔注射等量等渗的生理盐水。主要观察指标：以四甲基偶氮唑盐比色法检测利鲁唑对血管内皮生长因子诱导的人脐静脉内皮细胞增殖的影响。以组织切片苏木精一伊红染色观察并计数突破视网膜内界膜的血管内皮细胞核数目。以SABC法免疫组织化学染色观察视网膜组织中蛋白激酶C—BII及血管内皮生长因子的表达。结果：利鲁唑在0．1-10umol／L浓度范围内，可抑制培养的人脐静脉内皮细胞增殖，且呈剂量效应依赖关系。高氧模型组视网膜组织切片上见较多突破视网膜内界膜的血管内皮细胞核，治疗组突破视网膜内界膜的内皮细胞核数目明显减少（P〈0．01），免疫组织化学染色检测高氧模型组小鼠视网膜蛋白激酶C—βⅡ及血管内皮生长因子表达比正常对照组明显增强，治疗组比模型组明显减弱。结论：利鲁唑腹腔注射能抑制氧致视网膜新生血管的形成，对视网膜蛋白激酶C—βⅡ及血管内皮生长因子的表达有抑制作用，利鲁唑对体外培养人脐静脉内皮细胞增殖的抑制作用与其抑制蛋白激酶C-βⅡ的活性进而抑制血管生长因子的表达有关。

BACKGROUND： Studies show that riluzole can greatly inhibit protein kinase C- βⅡ in mediating vascular endothelial cell growth factor effects on endothelial cells, so we presume it can be used in inhibiting retinal neovascularization. OBJECTIVE： To explore the inhibitory effects of riluzole on the proliferation of human umbilical vein endothelial cells and retinal neovascularization, and investigate potential mechanism of its effects. DESIGN, TIME AND SETTING： Single-sample observation and randomized controlled animal trial were performed at the Animal Laboratory and Basic Study Laboratory of Chongqing University of Medical Sciences from August to December 200% MATERIALS： Thirty 3-day-old healthy C57BL/6 mice （60 eyes）, irrespective of gender, were selected to model proliferative retinopathy induced by hyperoxia. Raw material of riluzole was product of Venturepharm Laboratories Limited, Beijing （No. 060630, content ≥98.5%）. METHODS： Human umbilical vein endothelial cells were cultured, and randomly divided into 3 groups. The mice in hyperoxia model group and riluzole group were modeled, and riluzole group was additionally intraperitoneally injected 10 mg/kg per day riluzole; control group was intraperitoneally injected normal saline. MAIN OUTCOME MEASURES： Effect of riluzole on proliferation of endothelial cell proliferation induced by vascular endothelial growth factor was observed using MTT assay; The inhibitory effects of riluzole on retinal neovascularization were evaluated by counting the endotheliocyte nuclei of new vessels beyond the inner limiting membrane in sections with HE staining. The expression of retinal protein kinase C- βⅡ and vascular endothelial growth factor were detected with SABC immunohistochemistry. RESULTS： The riluzole of 0.1-10 u mol/L inhibited proliferation of human umbilical vein endothelial cells in a dose-effect dependent manner. The number of the endotheliocyte nuclei of new vessels beyond the inner limiting membrane was obviously less in eyes of normal control and riluzole groups than in the hyperoxia group （P 〈 0.01）. Immunohistochemistry of retinal sections revealed protein kinase C- βⅡ and vascular endothelial growth factor were overexpressed in the retina of the hyperoxia group compared with control group, while the expressions in riluzole group were significantly reduced. CONCLUSION： Retinal neovascularization is inhibited by intraperitoneal injections of riluzole, and the protein kinase C- βⅡ and vascular endothelial growth factor expression in retina is partially inhibited. The mechanism of inhibitory effect of riluzole may contribute to inhibiting the activity of protein kinase C- βⅡ and thus the expression of some important growth factor.