Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制

Inhibitory effects and mechanism of Arresten on retinal neovascularization in oxygen-induced retinopathy mice

目的探讨Arresten在氧诱导小鼠视网膜新生血管形成中的抑制作用及机制。方法选取48只出生后7 d健康清洁级C57BL/6J幼鼠,随机分为氧诱导视网膜病变(oxygen induced retinopathy,OIR)组、OIR+Arresten组和常氧组,每组16只。OIR+Arresten组及OIR组幼鼠在体积分数为(75±2)%的高氧环境下饲养5 d,然后转移至正常氧气环境中饲养5 d,其中OIR+Arresten组幼鼠于高氧饲养刚结束时,给予双眼玻璃体内注射Arresten蛋白。常氧组幼鼠则在常规氧气环境中连续饲养10 d。在鼠龄17 d时,各组取6只幼鼠经股静脉注射异硫氰酸葡聚糖溶液处死并摘出眼球,视网膜铺片后观察视网膜的血管形态、计算无灌注区面积。同时对小鼠眼球视网膜行石蜡切片和HE染色,计算侵入氉玻璃体内血管内皮细胞核的数量。同时,通过细胞培养实验,利用MTT法检测Arresten蛋白对人血管内皮细胞增殖的抑制作用。结果视网膜铺片结果显示,常氧组、OIR组、OIR+Arresten组视网膜无灌注区相对面积分别为(2. 35±1. 62)%、(57. 28±9. 36)%和(20. 38±8. 69)%,三组间总体比较,差异有统计学意义(F=18.732,P <0.05),且OIR组无灌注区相对面积显著高于OIR+Arresten组,差异有统计学意义(P <0. 05)。常氧组小鼠视网膜的内界膜结构仍保持平滑完整,未发现有新生血管侵入玻璃体内。OIR组和OIR+Arresten组每张切片上血管内皮细胞核的数量分别为(15. 18±4. 83)个、(7. 33±3. 88)个,其中OIR+Arresten组侵入玻璃体内新生血管内皮细胞核的数量显著低于OIR组,差异有统计学意义(P <0.05)。Arresten蛋白对人脐静脉内皮细胞增殖的抑制率随着其浓度的升高而增加,但当Arresten蛋白浓度达到1000μg·L^(-1)时,人脐静脉内皮细胞的增殖抑制率达到顶峰,为(58. 00±0. 65)%。结论 Arresten蛋白能够抑制氧诱导的小鼠视网膜新生血管形成,通过抑制血管内皮细胞增殖来抑制新生血管形成。

Objective To explore the inhibitory effect and the mechanism of Arresten protein on oxygen-induced retinal neovascularization in mice.Methods At postnatal 7 days(P7),forty-eight C57BL/6J pups were randomly divided into OIR group,OIR+Arresten group and normoxic group,there were 16 pups in each group.The pups in OIR group and OIR+Arresten group were exposed to high oxygen(75±2)%for 5 days.At P12,the pups in OIR+Arresten group were injected intravitreous with Arresten protein,then they were fed under normoxic condition for 5 days.The pups in normoxic group were fed under normoxic condition for 10 days.Six mice in each group were injected with FITC-dextrana via femoral vein at P17,the retinal vascular morphology was observed and the area of noperfusion was calculated.At the same time,paraffin sections and HE staining were performed on the mice retina,the average number of pre-retinal vascular endothelial nuclei were counted.The effect of Arresten protein on the proliferation of human vascular endothelial cells line was detected by MTT assay.Results FITC-dextran labeled retinal whole mounts showed that the relative area of noperfusion area in the normoxic,OIR group and OIR+Arresten were(2.35±1.62)%,(57.28±9.36)%and(20.38±8.69)%,respectively.The OIR group was higher than OIR+Arresten group,there was a statistical significance(P<0.05).The average number of endothelial nuclei in each section of the three groups was 15.18±4.83 and 7.33±3.88,respectively.The number of neovascularized endothelial nuclei in OIR+Arresten group was significantly lower than that in OIR group,and the inhibitory rate of Arresten protein on the proliferation of HUVEC cells were increased with the increase of Arresten protein concentration,but when the concentration of Arresten protein increase to 1000μg·L^-1,the proliferation inhibition rate of HUVEC cells arrived at its peak.Conclusion Arresten can inhibits retinal neovascularization by oxygen-induced via inhibiting the proliferation of vascular endothelial cells in mice.