谷氨酸受体相互作用蛋白对缺氧缺糖条件下少突胶质前体细胞凋亡的影响

Effects of AMPA receptor regulatory protein on the apoptosis of oligodendrocyte precursor cells under hypoxia and hypoglycemia conditions

目的观察缺氧缺糖条件下谷氨酸(AMPA)受体相互作用蛋白(GRIPs)表达的变化及对少突胶质前体细胞(OPCs)凋亡的影响,探讨GRIPs在AMPA受体兴奋性毒性中的作用。方法OPCs分为对照组、缺氧缺糖60 min组和缺氧缺糖120 min组,通过实时荧光定量PCR及蛋白印迹法(Western blot)检测缺氧缺糖条件下GRIP1、GRIP2 mRNA和蛋白的表达情况;再次将OPCs分为空白对照组、OPCs+缺氧缺糖组、OPCs+环磷酸腺苷(cAMP)+缺氧缺糖组、OPCs+cAMP+缺氧缺糖+GRIP1小干扰RNA(siRNA)组、OPCs+cAMP+缺氧缺糖+GRIP1 siRNA阴性对照组、OPCs+cAMP+缺氧缺糖+GRIP2 siRNA组、OPCs+cAMP+缺氧缺糖+GRIP2 siRNA阴性对照组,TUNEL试剂盒检测各组细胞凋亡情况,Fluo-4荧光探针观察胞内游离钙变化。结果缺氧缺糖引起OPCs损伤,光学显微镜下显示细胞轮廓不清晰,胞体回缩。缺氧缺糖60 min后GRIP1(1.233±0.060比1.003±0.079,P<0.05)和GRIP2(1.396±0.069比1.001±0.037,P<0.05)表达明显高于对照组,且缺氧缺糖时间越长,GRIP1(1.416±0.064比1.233±0.060,P<0.01)和GRIP2(1.680±0.018比1.396±0.069,P<0.01)表达水平越高。RNAi分别下调GRIP1和GRIP2,细胞内游离钙离子水平降低(0.054±0.003比0.074±0.003,P<0.01;0.060±0.003比0.074±0.003,P<0.01),细胞凋亡率下降[(20.703±3.882)%比(11.470±1.679)%,P<0.05;(19.070±1.106)%比(14.448±0.849)%,P<0.01]。结论GRIP1和GRIP2参与缺氧缺糖引起的OPCs损伤,其可能通过调节Ca^2+通透性触发AMPA受体介导的兴奋性毒性发挥作用。

Objective To observe the variation of glutamate(AMPA)receptor interacting protein(GRIPs)and apoptosis of oligodendrocyte precursor cells(OPCs)under oxygen glucose deprivation(OGD)condition,so as to explore the role of GRIPs in AMPA receptor-induced excitotoxic injury.Methods OPCs were divided into control group,60 min OGD group and 120 min OGD group.Real-time polymerase chain reaction(PCR)and Western blot were used to detect the mRNA and protein expressions of GRIP1 and GRIP2 under OGD conditions.OPCs were divided into blank control group,OPCs+OGD group,OPCs+cyclic adenosine monophosphate(cAMP)+OGD group,OPCs+cAMP+OGD+GRIP1 small interfering RNA(siRNA)group,OPCs+cAMP+OGD+GRIP1 siRNA negative control group,OPCs+cAMP+OGD+GRIP2 siRNA group,OPCs+cAMP+OGD+GRIP2 siRNA negative control group again,and terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL)kit was used to detect the apoptosis of each group.Fluo-4 fluorescent probe was used to measure the changes of intracellular free calcium.Results OGD caused damage to OPCs,and the light microscope showed that the cell contour was not clear and the cell body retracts.The expressions of GRIP1(1.233±0.060 vs.1.003±0.079,P<0.05)and GRIP2(1.396±0.069 vs.1.001±0.037,P<0.05)were significantly higher than those in control group after 60 min of OGD was,and the longer the period of OGD,the higher the expression levels of GRIP1(1.416±0.064 vs.1.233±0.060,P<0.01)and GRIP2(1.680±0.018 vs.1.396±0.069,P<0.01)were.When GRIP1 and GRIP2 were down-regulated,the level of intracellular free calcium ion decreased(0.054±0.003 vs.0.074±0.003,P<0.01;0.060±0.003 vs.0.074±0.003,P<0.01),and the apoptosis rate decreased as well[(20.703±3.882)%vs.(11.470±1.679)%,P<0.05;(19.070±1.106)%vs.(14.448±0.849)%,P<0.01].Conclusions GRIP1 and GRIP2 are involved in the damage of OPCs that are caused by OGD,which may trigger AMPA receptor-mediated excitotoxicity by regulating Ca^2+permeability.