低氧对小鼠RGC-5细胞中膜联蛋白A2表达的抑制作用  

Restrain of hypoxia on the expression of annexin A2 in mouse RGC-5 cells

作　　者：闫琳[1] 杨侠[1] 王龙梅 董晓光[1] 徐海峰[1] 

机构地区：[1]山东省眼科研究所 山东省眼科学重点实验室,青岛266071 [2]日照市中医院,276800

出　　处：《中华实验眼科杂志》2015年第12期1077-1082,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：山东省自然科学基金项目（2011ZRC03040）

摘　　要：背景研究已证实，缺氧是诱导视网膜新生血管形成的主要因素，与多种眼科病变的发生有关，且低氧与视网膜神经节细胞（RGCs）的变性也密切关联，但对视网膜神经组织病变的研究较少。目的观察低氧对RGCs中ANXA2表达的影响，探讨低氧对RGCs的损伤机制。方法用含体积分数10％胎牛血清的高糖培养液对小鼠RGC-5细胞株进行培养，采用免疫荧光技术检测RGCs特异表面抗原Thy-1的表达。分别采用含CoCl2终质量浓度为50、100、200、300μmol／L的培养液培养RGC-5细胞，并分别于培养后12、24、48h采用细胞计数试剂盒8（CCK-8）法检测细胞吸光度（A）值，以不含CoCl2培养液培养RGC-5细胞作为正常对照。以含终浓度为100μmol／LCoCl2培养液培养RGC-5细胞以建立低氧细胞模型，将模型细胞分为低氧3h组、低氧6h组、低氧12h组、低氧24h组，正常培养的细胞作为正常对照组。各组细胞行Hoeehst 33342荧光染色，观察RGC-5细胞核形态的变化；采用实时定量逆转录PCR检测各组细胞中ANXA2 mRNA的相对表达量；采用Western blot法检测细胞中ANXA2蛋白的相对表达量；采用免疫荧光法观察ANXA2在细胞中表达的分布。结果体外培养的RGC-5细胞呈梭形或多边形，Thy-1反应阳性。与正常对照组比较，50和100μmol／LCoCl2组各时间点细胞A值的差异均无统计学意义（均P〉0．05）；200和300μmol／L CoCl2组各时间点细胞A值均明显低于正常对照组，差异有统计学意义（均P〈0．05）。正常对照组细胞核Hoeehst 33342染色未见致密的亮蓝色荧光，低氧3h组、低氧6h组、低氧12组和低氧24组细胞核内均可见致密的亮蓝色强荧光。低氧3h组、低氧6h组、低氧12h组和低氧24h组RGC-5细胞中ANXA2 mRNA的相对表达量分别为0．80±0．14、0．67±0．33、0．49±0．17、0．39±0．02，均明显低于正常对照组，总体比较差异有统计学意义（F=434．354�Background Hypoxia is the main factor of retinal neovascularization and is closely associated with retinal ganglial cells （RGCs） degeneration. However, the study of retinal neural tissue lesions is rare. Objective This study was to investigate the influence of hypoxia environment on the expression of annexin A2 （ANXA2） in mouse RGC-5 cells and explore the mechanism of RGCs damage induced by hypoxia. Methods Immortalized mouse RGC-5 cells were cultured in high glucose DMEM with 10% fetal bovine serum. The cells were identified by detecting the expression of Thy-1 ,a specific biomarker of RGCs. CoCl2 was added into the medium at the final concentrations of 50,100,200 and 300 μmol/L, and the cells without CoCl2 served as the control group. Cell viability （absorbance） was assayed by cell counting kit-8 （CCK-8） method in 12,24 and 48 hours after addition of CoCl2. The hypoxic cell models were established in DMEM with 100 μmol/L CoCl2 and divided into the hypoxic 3- hour group, hypoxic 6-hour group, hypoxic 12-hour group and hypoxic 24-hour group, with the normal cultured cells as the normal control group. Apoptotic cells were determined by using hoechst 33342 stain. The expression levels of ANXA2 mRNA and protein in the cells were detected by real-time quantification PCR and Western blot,respectively. The expression and location of ANXA2 in the cells were examined by using immunofluorescence technique. Results The cultured ceils grew well and showed the fusiform and polygonal shape,with positive expression of Thy-1 protein. Compared with the normal control group, the viabilities of the ceils were insignificantly changed in the 50 μmol/L CoCl2 group and 100 μmol/L CoCl2 group （all at P〉0.05 ） , but the cell viabilities were significantly reduced in the 200 μmol/L CoCl2 group and 300 μmol/L CoCl2 group in various time points （ all at P 〈 0.05 ）. Hoechst 33342 staining showed that the apoptotic cells with nuclear condensation and high green fluorescence intensity were obtained in the hyp

关 键 词：膜联蛋白A2 视网膜神经节细胞 缺氧 细胞凋亡 

分 类 号：R774.1[医药卫生—眼科]