低氧对小鼠视网膜神经节细胞-5中根蛋白表达的影响  

Effects of hypoxia on radixin expression in retinal ganglion cell-5 of mice

作　　者：闫琳[1,2] 王龙梅[1,2] 杨侠[2,3] 董晓光[2,3] 徐海峰[2,3] 

机构地区：[1]潍坊医学院 [2]山东省眼科研究所,山东省潍坊市261053 [3]青岛眼科医院,山东省青岛市266071

出　　处：《眼科新进展》2014年第5期433-437,共5页Recent Advances in Ophthalmology

摘　　要：目的观察低氧对视网膜神经节细胞(retinal ganglion cells,RGC)-5中根蛋白(radixin,RDX)表达的影响,探讨低氧损伤中RGC的损伤机制和可能的保护途径。方法常规培养RGC-5,用RGC特异表面标记性抗原Thy-1鉴定;CoCl2法建立低氧模型,以含终浓度为100μmol·L-1CoCl2培养基培养RGC-5 3 h、6 h、12 h、24 h、48 h,采用Real-time RT-PCR和Western blot检测RDX mRNA及其蛋白在RGC-5中的表达情况,采用免疫荧光观察RDX在RGC-5细胞中的表达及定位。结果体外培养的RGC-5呈梭形或多边形,特异性表面标记性抗原Thy-1反应阳性。Real-time RT-PCR结果显示,低氧组RDX mRNA表达呈进行性增强,于低氧6 h达到高峰后开始下降;正常对照组RDX mRNA的相对表达量为1.00±0.11,低氧3 h、6 h、12 h、24 h、48 h分别为1.37±0.18、1.75±0.32、1.21±0.23、0.91±0.19、0.63±0.26,总体比较差异有统计学意义(F=813.717,P=0.000)。Western blot结果显示,不同培养条件下RGC-5细胞中RDX蛋白的总体比较差异有统计学意义(F=1.279,P=0.000)。低氧3 h、6 h、12 h、24 h组RDX/GAPDH A值均高于正常对照组,差异均有统计学意义(t=55.301,P=0.000;t=139.986,P=0.000;t=37.552,P=0.000;t=4.183,P=0.014);低氧48 h组RDX/GAPDH A值(0.24±0.03)低于正常对照组(0.33±0.02),差异有统计学意义(t=38.889,P=0.000)。在低氧组内不同时间点比较,RDX/GAPDH A值随时间的延长呈逐渐上升趋势,于低氧6 h达高峰,然后开始下降,差异有统计学意义(P<0.05)。免疫荧光观察发现RDX蛋白阳性表达在RGC-5细胞中,与正常对照组相比,低氧组RDX的表达先增强(6h)后减弱。结论低氧条件下小鼠RGC-5细胞中RDX表达水平先升高后降低,可能与视网膜新生血管疾病中RGC凋亡相关。Objective To investigate the effects of hypoxia on radixin （RDX） expression in retinal ganglion cells （RGC）-5, and discuss the mechanism and possible protective pathway of RGC damage induced by hypoxia. Methods RGC-5 were rou- tinely incubated and indentified with Thy-1. Then RGC-5 were cultured in DMEM for 3 hours,5 hours, 12 hours, 24 hours and 48 hours, the culture medium contained 100 μmol · L-1 cobalt chloride to establish the hypoxia model. Reverse transcription poly- merase chain reaction （RT-PCR） and Western blot were used to evaluate the expres- sion of RDX mRNA and protein in RGC-5 at different time points. The expression and location of RDX in RGC-5 were investigated by immunocytochemistry, Results Cul- tured RGC-5 with fusiform and polygon shape positively expressed Thy-1 antigen. RT- PCR showed that RDX mRNA expression levels in RGC-5 in hypoxia group gradually in- creased and peaked at 5 hours, and later began to decline. The expression levels of RDX mRNA at 0 hour,3 hours,5 hours, 12 hours,24 hours and 48 hours were 1.00 ± 0. 11, 1.37 ± 0.18,1.75 ± 0.32,1.21± 0.23,0.91 ± 0.19 and 0.53 ±0.25, respectively, and there was significant difference among 6 groups （ F = 813.717,P = 0. （D0）. Western blot deter- mined that the expression level （A value） of RDX protein in RGC-5 was significantly different in each culture condition （ F = 1. 279, P = 0.000）. The A value of the RDX/GAP- DH at 3 hours,5 hours,12 hours and 24 hours in hypoxia group were higher than those in control group （ t = 55. 301 ,P = 0. 000; t = 139.985 ,P = 0. 000 ; t = 37.552 ,P = 0.000; t = 4. 183 ,P = 0.014）, but at 48 hours these were lower than control group （t = 38.889 ,P = 0. 000）. The A value of the RDX/GAPDH was presented rising trend as the extension of time in hypoxia group at different time points, and peaked at 5 hours, but later de- creased （ P 〈 0. 05 ）. Immunocytochemistry showed that the RDX were expressed in RGC-5, and the expression of RDX in hypoxia groups were strengthened

关 键 词：根蛋白 视网膜神经节细胞-5 缺氧 细胞活力 

分 类 号：R775[医药卫生—眼科]