糖原合酶激酶3β对体外加压培养下的视网膜神经节细胞的作用

摘要

目的 研究体外加压培养的视网膜神经节细胞(RGCs)中糖原合酶激酶3β(GSK3)活性变化及其对RGCs存活的影响。方法 实验研究。混合培养大鼠RGCs，分为RGCs无加压对照组、单纯RGCs加压组（60 mmHg压力下培养24h）、RGCs加压+10 mmol/L氯化锂（Licl，GSK3β抑制剂）组和RGCs加压+20 mmol/L Licl组。采用免疫荧光法检测RGCs中磷酸化丝氨酸9位点GSK3β(pS9-GSK3β）的表达情况；以MTT法检测吸光度(OD)值反映RGCs活性。对数据进行单因素方差分析。结果 加压组pS9-GSK3β表达的荧光密度平均光密度（MOD）值为0.057±0.012，较对照组(0.085±0.023)低，而加压+10 mmol/L Licl组和加压+20 mmol/L Licl组pS9-GSK3β表达的荧光密度(分别为0.081±0.016和0.099±0.024)均较加压组大，且随着Licl浓度的增加，pS9-GSK3β表达的荧光密度增大。MTT结果显示，4个实验组的OD值差异有统计学意义（F=14.539，P＜0.05）。加压组OD值(0.109±0.016)较对照组(0.135±0.015)低(P＜0.05)，细胞活性较低；而加压+20 mmol/L Licl组中OD值(0.159±0.014)较加压组高(P＜0.05)，细胞活性较高。结论 压力培养下，视网膜神经节细胞中pS9-GSK3β表达减少，提示GSK3β活性增强，RGCs活性降低。Licl作为GSK3β的抑制剂，可能对RGCs有保护作用。%Objective To investigate the activity of glycogen synthase kinase-3β (GSK3β) on retinal ganglion cells (RGCs) under pressure, and the effect of GSK3β on the survival of RGCs.Methods This was an experimental study. Rat RGCs were mixed cultured in vitro and then divided into RGCs control group, RGCs pressure only group (60 mmHg of pressure cultivated for 24 hours),RGCs pressure group treated with 10 mmol/L lithium chloride (Licl, inhibitor of GSK3β) and RGCs pressure group treated with 20 mmol/L lithium chloride. Phospho-Ser9-GSK3β (pS9-GSK3β) was observed by immunocytochemical staining. Meanwhile, MTT method test optical density (OD) value to reflect the activity of RGCs of each group. The data for all groups were analyzed by an one-way ANOVA. Results Compared to the RGCs control group [mean optic density (MOD) was 0.085±0.023], the pressure only group expressed less pS9-GSK3β (0.057±0.012). However, both Licl treated groups, expressed more pS9-GSK3β (0.081±0.016 and 0.099±0.024) than the pressure only group.Meanwhile, as the concentration of Licl increased, the higher levels pS9-GSK3β were expressed. Moreover, MTT results showed that the difference of OD value in four groups had statistical significance (F=14.539, P＜0.05). OD value of pressure only group (0.109±0.016) was significantly lower than control group (0.135±).015) (P＜0.05), and RGCs viability was reduced. Compared to the OD value of the 20 mmol/L Licl treated group (0.159±0.014), the pressure only group showed lower OD value (P＜0.05), and the viability of RGCs increased. Conclusion The reduction of pS9-GSK3β in RGCs under pressure prompts GSK3β to be activated, so the viability of RGCs is lower than that of normal RGCs.However, Licl, as the inhibitor of GSK3β, maybe have the ability to protect RGCs from apoptosis.