TRPC3参与糖氧剥夺致培养的少突胶质细胞凋亡

Oxygen Glucose Deprivation-induced Apoptosis in Oligodendrocytes Through TRPC3

目的:研究瞬时受体电位通道蛋白3(transient receptor potential channel 3,TRPC3)是否参与糖氧剥夺(oxygen glucose deprivation,OGD)少突胶质细胞的凋亡。方法:以原代培养的新生SD大鼠少突胶质细胞为对照组,以OGD2h的少突胶质细胞为模型组,将模型组+Pyr3阻断设为处理组。采用蛋白质免疫印迹法检测TRPC3蛋白的表达水平,MTT试剂盒比色法检测各组细胞存活率,Annexin V-FITC试剂盒染色后经流式细胞仪检测细胞凋亡,fluo-3染色后经流式细胞仪和激光共聚焦显微镜测定胞内游离钙的变化。结果:少突胶质细胞A2B5和MBP特异性标记阳性,细胞纯度可达到95%以上;少突胶质细胞OGD2h成功建立OGD模型;OGD组TRPC3蛋白表达增加;OGD组细胞活性为(54.34±6.55)%,凋亡率为(24.24±0.86)%,与对照组有显著差异(P<0.05),Pyr处理组细胞存活率为(72.26±5.41)%,凋亡率为(14.82±0.28)%,与OGD组有显著差异(P<0.05);OGD组胞内游离钙浓度显著升高,而Pyr3阻断以后可以部分抑制其升高。结论:TRPC3表达增加介导胞内游离钙离子水平的升高可能是OGD少突胶质细胞凋亡的重要原因之一。

Objective： To explore whether transient receptor potential canonical 3 （TRPC3） plays a role in the apoptosis of oligodendrocytes induced by oxygen glucose deprivation（OGD）.Methods： Control group were established by primary cultured oligodendrocytes of SD rats,model group were established by primary cultured oligodendrocytes of SD rats which exposed to OGD2h,treated group were established by model group＋ Pyr3.Western blotting were used to detect the expression of TRPC3 at protein levels.The viability of the oligodendrocytes was determined by MTT assay,and the apoptosis of oligodendrocytes were measured by flow cytometry after AnnexinV-FITC staining,intracellular Ca2＋ concentration were measured by flow cytometry and Laser Confocal Scamfing Microscope（LCSM）.Results： Specific markers of A2B5 and MBP in oligodendrocytes were positive and the purification is more than 95%,OGD model was established successfully.A significant increase in the expression of TRPC3 in cultured oligodendrocytes.The viability of OGD oligodendrocytes （54.34±6.55）% was obviously decreased and the apoptosis rate was increased （24.24±0.86）% when compared with the control cells （P〈0.05）.Pyr3 significantly increased the survival rate （72.26±5.41）% and decreased the apoptosis rate （14.82±0.28）% and partially suppressed Ca2＋ in oligodendrocytes （P〈0.05）.Conclusion： The increased expression of TRPC3 which mediated intracellular calcium level may be one of the important reasons for OGD oligodendrocyte apoptosis.