A53T α-synuclein抑制SH-SY5Y细胞2型囊泡单胺转运体的表达

A53T α-synuclein decreases the expression of type 2 vesicular monoamine transporter in neuroblastoma SH-SY5Y cells

目的研究稳定表达A53Tα-synuclein对神经母细胞瘤SH-SY5Y细胞中2型囊泡单胺转运体(VMAT2)蛋白表达的影响。方法构建A53Tα-synuclein真核表达载体,用脂质体lipofectamineTM2000转染SHSY5Y细胞,经G418筛选获得稳定转染单克隆细胞株;分别采用Western blotting及DCFH-DA染色检测细胞系A53Tα-synuclein过表达对VMAT2蛋白的表达及细胞内活性氧类物质(ROS)水平的影响。结果 Western blotting结果显示VMAT2的表达在稳定表达A53Tα-synuclein的细胞系中明显降低。DCFH-DA染色结果显示稳定表达A53Tα-synuclein细胞系内DCF signal(507.3±7.1)较对照组(410.7±10.5)明显增多(P<0.05)。结论 A53Tα-synuclein能够通过抑制VMAT2的表达,增加细胞内的ROS水平,在帕金森病的发病过程中发挥重要作用。

Objective To investigate the effect of A53 T α-synuclein on the expression of type 2 vesicular monoamine transporter（ VMAT2） in neuronblastoma SH-SY5 Y cells stably expressing A53 T α-synuclein. Methods A53 T α-synuclein eukaryotic plasmid was constructed by transfection of the SH-SY5 Y cells using LipofectamineTM2000,and a stable transfected monoclonal cell line was selected by G418. Western blotting and DCFH-DA staining were used to detect the effect of A53 T α-synuclein overexpression on the expression of VMAT2 protein and level of reactive oxygen species（ ROS）. Results Western blotting showed that compared with the control group,the expression of VMAT2 protein was significantly decreased,and DCFH-DA staining showed that DCF signal was significantly increased（ 507. 3 ± 7. 1）than that in the cell line stably expressing A53 T α-synuclein（ 410. 7 ± 10. 5）（ P〈0. 05）. Conclusions A53 T α-synuclein can increase the intracellular ROS level by inhibiting the expression of VMAT2,thereby playing an important role in the pathogenesis of Parkinson＇s disease.