活性氧簇响应型纳米药物对活体小鼠角膜新生血管形成抑制作用的评估  

In vivo investigation of the effectiveness of reactive oxygen species-responsive nanomedicine in suppressing corneal neovascularization

作　　者：刘安琪[1] 梁春菁 汪铭[2] 王丽强[1] 黄一飞[1] Liu Anqi;Liang Chunjing;Wang Ming;Wang Liqiang;Huang Yifei(Department of Ophthalmology,Liberation Army General Hospital,Beijing 100853,China;Institute of Chemistry,the Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]解放军总医院眼科,北京100853 [2]中国科学院化学研究所,北京100190

出　　处：《中华实验眼科杂志》2020年第2期85-92,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家重点研发计划项目(2017YFA0103204);国家自然科学基金项目(81770887)。

摘　　要：目的评估活性氧簇(ROS)响应型纳米药物对活体小鼠角膜新生血管(CNV)形成的影响。方法Michael加成反应合成含有缩硫酮键的载血管内皮生长因子小干扰RNA(siVEGF)的ROS响应型纳米药物(ROS-TK-5/siVEGF)。琼脂糖凝胶电泳测定纳米药物在高ROS环境中的siVEGF累计释放量。选取39只6~8周龄VEGFR2-luc-KI转基因荧光标记小鼠,采用随机数字表法将其中30只小鼠分为正常对照组、PBS对照组、ROS-TK-5/NC组、ROS-TK-5/siVEGF组和雷珠单抗组,每组6只,采用NaOH滤纸贴附角膜中央的方法构建小鼠右眼CNV模型,正常对照组不做处理;应用随机数字表法将另外9只小鼠分为正常对照组、建模后7 d组和建模后14 d组,每组3只小鼠,采用二氢乙啶(DHE)染色法测定小鼠角膜碱烧伤后角膜组织内ROS含量。裂隙灯显微镜眼前节照相与小动物活体成像系统(IVIS)观察小鼠角膜碱烧伤后7、14、21 d应用纳米药物对CNV形成的影响。结果在无ROS环境中,仅5%~10%的siVEGF从纳米复合物中释放出。与10 mmol/L H2O2共孵育10 h后,约70%的siVEGF从纳米药物中释放。建模后7 d和14 d,角膜基质层相对荧光强度分别为5.403±0.306和2.930±0.255,较正常对照组的1.003±0.015明显增加,差异均有统计学意义(均P<0.05)。各组建模后不同时间CNV面积比较,差异均有统计学意义(F分组=49.855,P<0.01;F时间=65.556,P<0.01),其中建模后7 d和14 d,ROS-TK-5/siVEGF组和雷珠单抗组的CNV面积较PBS对照组和ROS-TK-5/NC组明显减小,ROS-TK-5/siVEGF组CNV面积较雷珠单抗组明显减小,差异均有统计学意义(均P<0.05);建模后21 d,ROS-TK-5/siVEGF组和雷珠单抗组的CNV面积较PBS对照组及ROS-TK-5/NC组明显减小,差异均有统计学意义(均P<0.05)。各组小鼠建模后不同时间小鼠角膜的荧光信号强度比较,差异均有统计学意义(F分组=27.193,P=0.003;F时间=51.062,P<0.01),其中建模后7 d和14 d,ROS-TK-5/siVEGF组和雷珠单抗组的角膜荧光信号强Objective To evaluate the effectiveness of reactive oxygen species(ROS)-responsive nanomedicine in suppressing corneal neovascularization(CNV)in vivo.Methods ROS-responsive nanomedicine(ROS-TK-5/siVEGF),which consists of vascular endothelial growth factor(VEGF)small interfering RNA(siRNA)and thioketal linkage was synthesized by the Michael addition.The cumulative release of siVEGF from nanomedicine under oxidant conditions was assessed by agarose gel electrophoresis.Thirty-nine VEGFR2-luc-KI transgenic mice were used in this study,of which 30 mice were randomly divided into a normal control group,a PBS control group,an ROS-TK-5/NC group,an ROS-TK-5/siVEGF group,and a ranibizumab group,with 6 mice in each group.The ROS levels in the corneal tissue after alkali burning were tested by dihydroethidium(DHE)staining in the other 9 mice.In each group,alkali-burned mice were subconjunctivally injected with 10μl of a different formula every two days.The effectiveness of nanomedicine in attenuating CNV was evaluated by slit-lamp microscopy and an in vivo imaging system(IVIS)at 7,14,and 21 days after alkali burning.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology(ARVO)and the Guidelines of the Animal Experimental Committee of Liberation Army General Hospital.The study protocol was approved by the Ethics Committee of Liberation Army General Hospital(No.2018-X14-82).Results After treathrent with an aqueous solution without ROS,only 5%-10%of the siVEGF was released from the nanoparticles within 10 hours.In contrast,about 70%of the siVEGF was released from the nanoparticles after treatment with 10 mmol/L H2O2 within 10 hours.The relative fluorescent intensities in the corneal stromal layer at 7 days and 14 days after alkali burning were 5.403±0.306 and 2.930±0.255,respectively,which was significantly greater than those in the normal control group(1.003±0.015)(both at P<0.05).The CNV areas were statistically different among the four groups at various time

关 键 词：活体成像系统 碱烧伤 角膜新生血管 活性氧簇 纳米药物 

分 类 号：R77[医药卫生—眼科]