甲基苯丙胺对体外培养SH-SY5Y细胞线粒体膜电位、超微结构及Mfn1、Fis1蛋白表达的影响

Effects of METH on mitochondrial membrane potrntial,ultrastaucture and expression of Mfn1 and Fis1 protein in SH-SY5Y cells cultured in vitro

目的 评估甲基苯丙胺(METH)对体外培养的SH-SY5Y细胞线粒体膜电位(MMP)、线粒体超微结构及Mfn1、Fis1蛋白表达的影响。方法 SH-SY5Y细胞中加入METH(0.0、1.0、1.5、2.0 mmol·L^-1 ),培养3、6、12、24 h;JC-1法检测SH-SY5Y细胞MMP;透射电镜观察SH-SY5Y细胞线粒体超微结构;Western blot检测Mfn1、Fis1蛋白表达。结果 METH作用SH-SY5Y细胞3、6、12、24 h,与对照组比较,METH组细胞MMP红绿荧光比值明显降低( P <0.05),Mfn1蛋白表达降低( P <0.05),Fis1蛋白表达升高( P <0.05);METH作用SH-SY5Y细胞24 h时,透射电镜观察见未处理组细胞线粒体呈椭圆形棒状双层膜结构,线粒体嵴正常、清晰,METH组细胞线粒体椭圆形棒状结构被分裂成小球状结构,并发现线粒体自噬小体及自噬溶酶体。结论 METH可引起SH-SY5Y细胞MMP下降、线粒体超微结构改变及Mfn1、Fis1蛋白表达水平改变,其改变可能参与METH致神经细胞损伤的发病机制。

Aim To evaluate the influence of METH on MMP, mitochondrial ultrastructure, and the expression levels of mitochondrial proteins, Mfn1and Fis1, in human neuroblastoma SH-SY5Y cells in vitro .Methods A stable and feasible culture method of SH-SY5Y cells in vitro was established with different concentrations of METH(0.0,1.0,1.5 and2.0 mmol·L^-1 ), and for various periods of exposure for3, 6,12,24 h, the MMP of SH-SY5Y cells was stained by MMP assay kit(JC-1), the mitochondrial ultrastructure of SH-SY5Y cells exposed to METH was observed by transmission electron microscope, and the expression levels of Mfn1 and Fis1 proteins were detected by Western blot.Results Compared with control group for various periods of exposure(3,6,12,24 h), the red/green fluorescence ratios of MMP and the expression levels of Mfn1 protein decreased significantly in METH groups( P <0.05), while the expression levels of Fis1 protein increased significantly( P <0.05).SH-SY5Y cells were treated with METH for24 h prior to observation under transmission electron microscope(TEM).The mitochondria of SH-SY5Y cells in unprocessed group showed the oval, rodlike and double-layer membrane structure, along with clear normal mitochondrial cristae.However, the oval and rodlike structure of mitochondria in SH-SY5Y cells of METH treatment groups had been split into small ball structures.Moreover, mitochondrial autophagosome and autophagic lysosome could also be found.Conclusions METH could induce a decrease in MMP, mitochondrial ultrastructural changes, and changes in the expression levels of Mfn1 and Fis1 in SH-SY5Y cells, which might be associated with nerve cell damage caused by METH.