低氧对小鼠视网膜神经节细胞-5中根蛋白表达的影响

发布时间：2018-07-10 04:11

本文选题：根蛋白 + 视网膜神经节细胞-　；参考：《眼科新进展》2014年05期

【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(P0.05)。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。
[Abstract]:Objective to observe the effect of hypoxia on the expression of root protein (RDX) in (retinal ganglion cells of retinal ganglion cells (RGCs) -5, and to explore the mechanism and possible protective mechanisms of RGCs in hypoxic injury. Methods RGC-5 was cultured routinely and RGC-specific surface marker antigen Thy-1 was used to identify hypoxia model. RGC-53 h ~ (6) h ~ (12) ~ (24) h ~ (24) h ~ 48 h culture medium containing 100 渭 mol L ~ (-1) CoCl2. The expression of RDX mRNA and its protein in RGC-5 was detected by real-time RT-PCR and Western blot. The expression and localization of RDX in RGC-5 cells were observed by immunofluorescence. Results RGC-5 in vitro was fusiform or polygonal, and the expression of RDX mRNA in hypoxia group was increased progressively, and then decreased after 6 hours of hypoxia, and the specific surface marker antigen Thy-1 reaction was positive. Real-time RT-PCR showed that the expression of RDX mRNA in hypoxia group increased progressively. The relative expression of RDX mRNA was 1.00 卤0.11 in the control group and 1.37 卤0.18 卤1.75 卤0.32 卤0.23 卤0.63 卤0.26 in the control group, respectively. The results of Western blot showed that the RDX protein in RGC-5 cells cultured under different culture conditions was significantly different from that in the control group (F813.717P0. 000) (F1. 279P0. 000). The RDX / GAPDH A value in hypoxia group was significantly higher than that in normal control group (t = 55.301), and the difference was statistically significant (t ~ (55.301) P ~ (0.000) P ~ (0.000) P ~ (0.000) ~ (0.000) ~ (0.552) ~ (0.000) ~ (0.552) P ~ (0.000) P ~ (0.000) ~ (4) 183P0.014), and (0.24 卤0.03) (0.24 卤0.03) in hypoxia 48 h group was lower than that in normal control group (t ~ (38.889P ~ (0.000) (t ~ (38.889P ~ (0.000), the difference was statistically significant (t ~ (38.889P ~ (0.000). The value of RDX / GAPDH A increased gradually with the prolongation of time and reached its peak at 6 h after hypoxia and then began to decrease. The difference was statistically significant (P0.05). The positive expression of RDX protein in RGC-5 cells was observed by immunofluorescence. Compared with the control group, the expression of RDX in hypoxia group was increased at first (6 h) and then decreased. Conclusion the expression of RDX in RGC-5 cells increased first and then decreased under hypoxia, which may be related to the apoptosis of RGC in retinal neovascularization disease.
【作者单位】：潍坊医学院;山东省眼科研究所;青岛眼科医院;
【分类号】：R774.1

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