摘要
目的观察氧糖剥夺再灌注(OGD/R)后星形胶质细胞活化、损伤、凋亡、自噬水平的变化,探讨自噬在星形胶质细胞缺血性损伤中的作用。方法分离C57BL/6小鼠的星形胶质细胞,分为对照组和氧糖剥夺再灌注(OGD/R)组,OGD/R组构建OGD/R模型并分别于缺氧缺糖培养后3、6、12 h更换正常培养基,对照组不做特殊处理;采用Western blotting法检测细胞中的星形胶质细胞活化标志物胶质纤维酸性蛋白(GFAP)、Caspase-3、LC3B-Ⅱ,采用CCK-8实验检测并计算细胞存活率,采用二硝基苯肼法检测LDH漏出率,ELISA法检测细胞培养液中的TNF-α。分离C57BL/6小鼠的星形胶质细胞,分为对照组、3-MA对照组、OGD/R组、OGD/R+3-MA组;对照组不做特殊处理,OGD/R组、OGD/R+3-MA组建立OGD/R模型(缺氧缺糖培养后6 h更换正常培养基),3-MA对照组及OGD/R+3-MA组造模前加入1 mmol/L的3-MA;检测GFAP、细胞存活率、LDH漏出率、TNF-α水平、Caspase-3。结果OGD/R组不同处理时间细胞GFAP表达、LDH漏出率、TNF-α水平均高于对照组,细胞存活率低于对照组(P均<0.05);OGD/R组6 h、12 h时的Caspase-3/Pro-Caspase-3及LC3B-Ⅱ表达高于对照组(P均<0.05)。OGD/R+3-MA组细胞GFAP表达、LDH漏出率、TNF-α水平、Caspase-3/Pro-Caspase-3表达均低于OGD/R组,细胞存活率高于OGD/R组(P均<0.05)。结论OGD/R后星形胶质细胞活化和损伤增强,凋亡蛋白表达增加,自噬激活。自噬可能促进了星形胶质细胞的活化、损伤、凋亡,诱导星形胶质细胞缺血性损伤。
Objective To observe the changes of activation,injury,apoptosis and autophagy of oxygen-glucose deprivation/reoxygenation(OGD/R)astrocytes,and to explore the role of autophagy in the ischemic injury of astrocytes.Methods The astrocytes of C57BL/6 mice were isolated and divided into the control group and the OGD/R group.The astrocytes of the OGD/R group were used to make the OGD/R models and the normal medium was replaced at 3,6 and 12 h after hypoxic-glucose culture.The astrocytes of the control group received no special treatment.Western blotting was used to detect astrocyte activation markers glial fibrillary acidic portein(GFAP),Caspase-3,and LC3B-Ⅱ.CCK-8 assay was used to calculate cell viability.The dinitrophenylhydrazine method was used to detect the leak rate of LDH.ELISA was used to detect TNF-αin cell culture fluid.Then we isolated astrocytes from C57BL/6 mice and divided them into the control group,3-MA control group,OGD/R group,and OGD/R+3-MA group.No special treatment was given to the control group.The isolated astrocytes in the OGD/R group and OGD/R+3-MA group were used to establish the OGD/R models(normal medium was replaced 6 h after hypoxic and hypoglycemic culture),and the isolated astrocytes in the 3-MA group and OGD/R+3-MA group were added with 1 mmol/L of 3-MA before modeling,and then GFAP,cell survival rate,LDH leakage rate,TNF-αlevel and Caspase-3 were detected.Results GFAP expression,LDH leakage rate and TNF-αin the OGD/R group at different treatment time were all higher than those in the control group,and the cell survival rate was lower than that in the control group(all P<0.05).In the 6-hour and 12-hour OGD/R subgroups,Caspase-3/Pro-Caspase-3 and LC3B-Ⅱexpression levels were higher than those of the control group(all P<0.05).GFAP expression,LDH leakage rate,TNF-αlevel,Caspase-3/Pro-Caspase-3 expression in the OGD/R+3-MA group were all lower than those in the OGD/R group,and cell survival rate was higher than that in the OGD/R group(all P<0.05).Conclusions The activation and injury of OGD/R astrocytes are enhanced,the expression of apoptotic proteins increase,and autophagy is activated.Autophagy may promote the activation,injury,and apoptosis of astrocytes,and induce ischemic injury of astrocytes.
作者
胆迎
李晨
DAN Ying;LI Chen(Tianjin Fifth Central Hospital,Tianjin 300450,China)
出处
《山东医药》
CAS
2020年第8期48-51,共4页
Shandong Medical Journal
