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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 investigate whether transient receptor potential channel 3 (TRPC3) is involved in the apoptosis of oligodendrocytes in oxygen glucose deprivation (OGD). Methods: Primary cultured oligodendrocytes from SD rats were used as control group. OGD2h oligodendrocytes were used as model group, and model group + Pyr3 block was set as treatment group. The expression of TRPC3 protein was detected by Western blotting. Cell viability was detected by MTT kit colorimetric assay. Cell apoptosis was detected by flow cytometry after staining with Annexin V-FITC kit. Fluo-3 staining Cytometry and Laser Scanning Confocal Microscope for Determination of Intracellular Free Calcium. Results: The oligodendrocyte A2B5 and MBP were positive, the purity of which reached more than 95%. OGD model was successfully established in oligodendrocyte OGD2h. The expression of TRPC3 protein in OGD group was increased. The cell viability in OGD group was (54.34 ± 6.55) ), The apoptosis rate was (24.24 ± 0.86)%, which was significantly different from the control group (P <0.05). The cell survival rate was (72.26 ± 5.41)% and the apoptosis rate was (14.82 ± 0.28)% (P <0.05). The concentration of intracellular free calcium in OGD group was significantly increased, but was inhibited by Pyr3. CONCLUSION: Increased intracellular free calcium levels with increased expression of TRPC3 may be one of the important causes of apoptosis of OGD oligodendrocytes.