摘要
目的观察膜联蛋白A2(annexin A2,ANXA2)对糖基化终末产物牛血清白蛋白(advanced glycation bovine serum albu-min,AGE-BSA)培养的人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)骨架重塑、细胞迁移能力的影响,探讨ANXA2在糖尿病视网膜新生血管病变中的作用,为视网膜新生血管性疾病的防治寻找有效的分子靶点提供理论依据。方法用含AGE-BSA(0.2g·L-1)的高糖型DMEM培养液培养HUVEC,并用ANXA2 siRNA干扰AGE-BSA培养的HUVEC中ANXA2的表达;免疫沉淀及Western blot检测各组中ANXA2蛋白的表达;FITC-鬼笔环肽染色观察ANXA2对HUVEC骨架重塑的影响;划痕法观察ANXA2对HUVEC迁移能力的影响。结果空白对照组、阴性干扰组、阳性干扰组ANXA2蛋白的表达量分别为1.17±0.03、1.18±0.04、0.68±0.05,阳性干扰组与空白对照组之间差异有显著统计学意义(F=152.76,P=0.000);空白对照组、BSA组、AGE-BSA组、阴性干扰组、阳性干扰组处理24h后骨架染色显示:AGE-BSA组同空白对照组相比,细胞骨架应力纤维排布改变,而阳性干扰组同空白对照组骨架排布相仿;空白对照组、BSA组、AGE-BSA组、阴性干扰组、阳性干扰组处理24h后迁移入划痕的细胞数分别为153±12、143±16、192±8、176±6、142±14,AGE-BSA组与空白对照组之间、阳性干扰组与AGE-BSA组之间差异均有统计学意义(均为P<0.05);空白对照组、BSA组、AGE-BSA组处理24h后磷酸化ANXA2蛋白的表达量分别为0.94±0.57、0.94±0.32、1.12±0.95,AGE-BSA组与空白对照组之间差异有统计学意义(F=6.899,P<0.05)。结论 ANXA2参与AGE-BSA诱导的HUVEC细胞骨架重塑及细胞迁移能力改变,并且极可能通过其酪氨酸磷酸化形式来发挥中介作用,针对ANXA2生物学特性的深入研究可能为糖尿病视网膜病变的预防治疗提供新的思路及方法。
Objective To observe the effect of annexin A2（ANXA2）on cytoskeleton reconstruction and cells migration in human umbilical vein endothelial cells（HUVEC） induced by advanced glycation end products bovine serum albumin（AGE-BSA）,and study the role of ANXA2 in diabetic retinal neovascularization to provide the theory basis for founding the molecular target.Methods After the supplementation of AGE-BSA（200 g·L-1） in DMEM and the small interfering RNA（siRNA）-mediated knockdown of ANXA2 expression in HUVEC,the expression of ANXA2 protein was detected by immunoprecipitation and Western blot,the effect of ANXA2 on reconstruction of cytoskeleton was observed by immunofluorescence microscope with FITC-Phalloidin,and the effect of ANXA2 on HUVEC migration was observed by cell scratch.Results The expression of ANXA2 protein in untreated group,control siRNA group and ANXA2 siRNA group were 1.17±0.03,1.18±0.04 and 0.68±0.05,respectively,there was statistical difference between normal group and positive interfered group（F=152.76,P=0.000）.The cytoskeleton staining at 24 hours in control group,BSA group,AGE-BSA group,control siRNA group and ANXA2 siRNA group showed,compared with the control group,the stress fiber arrange of cytoskeleton in AGE-BSA group was changed,but which in positive interfered group was similar;The number of cells migrated to the scratch at 24 hours in control group,BSA group,AGE-BSA group,control siRNA group and ANXA2 siRNA group were 153±12,143±16,192±8,176±6,142±14,respectively,there was statistical difference between AGE-BSA group and control group,ANXA2 siRNA group（F=10.231,P0.05）;The values of phosphorylative ANXA2 protein expression at 24 hours in control group,BSA group and AGE-BSA group were 0.94±0.57,0.94±0.32 and 1.12±0.95,respectively,there was statistical difference between AGE-BSA group and control group（F=6.899,P0.05）.Conclusion ANXA2 take part in cytoskeleton reconstruction and HUVEC migration induced by AGE-BSA,which mostly may be through the way of ty
出处
《眼科新进展》
CAS
北大核心
2012年第4期322-326,共5页
Recent Advances in Ophthalmology
基金
山东省自然科学基金资助(编号:ZR2011HM012)~~
关键词
膜联蛋白A2
糖基化终末产物
细胞骨架
迁移
磷酸化
annexin A2
advanced glycation end products
actin cytoskeleton
migration
phosphorylation
