钙稳态失衡在1-甲基-4-苯基吡啶离子诱导的人神经母细胞瘤SH-SY5Y细胞凋亡中的作用  

Role of calcium dyshomeostasis in 1-methyl-4-phenylpyridinium ion-induced apoptosis of human neuroblastoma SH-SY5Y cells

作　　者：徐蕾[1] 李文军[1] 林丹苗[1] 张洪梅[1] 邹飞[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院职业卫生与职业医学系.广东广州510515

出　　处：《南方医科大学学报》2013年第4期479-485,共7页Journal of Southern Medical University

基　　金：国家自然科学基金(30971193)~~

摘　　要：目的研究钙稳态失衡在1-甲基-4-苯基吡啶离子(MPP+)诱导SH-SY5Y细胞凋亡中的作用。方法 MTT法检测细胞存活率;Hoechst 33342和Annexin V+PI染色检测MPP+诱导人神经神经母细胞瘤SH-SY5Y细胞的凋亡作用;Western blot检测PARP蛋白表达的变化;罗丹明123染色检测MPP+对SH-SY5Y细胞线粒体膜电位的影响;激光共聚焦显微镜检测MPP+对SH-SY5Y细胞胞质钙、线粒体钙和内质网钙浓度的影响以及线粒体钙通道抑制剂钌红与MPP+联合应用对线粒体游离钙浓度、胞质游离钙浓度的影响。结果 MPP+导致SH-SY5Y细胞存活率下降且具有剂量-反应关系、MPP+能导致SH-SY5Y细胞凋亡、MPP+导致SH-SY5Y细胞线粒体膜电位下降、MPP+使SH-SY5Y细胞胞质和内质网游离钙离子浓度下降而使线粒体游离钙离子浓度上升。钌红可对抗MPP+诱导的SH-SY5Y细胞凋亡、阻止线粒体膜电位下降,减少PARP的切割片段以及阻断线粒体游离钙浓度的上升。结论线粒体钙超载在MPP+诱导的SH-SY5Y细胞凋亡中起重要作用。MPP+诱导人神经母细胞瘤SH-SY5Y细胞的凋亡可能与细胞质、线粒体及内质网的钙稳态失衡有关。Objective To investigate the role of calcium dyshomeostasis in 1-methyl-4-phenylpyridinium ion（MPP＋）-induced apoptosis of human neuroblastoma SH-SY5Y cells.Methods The viability of SH-SY5Y cells exposed to varying concentrations of MPP ＋ was assessed using MTT colorimetric assay,and MPP ＋-induced cell apoptosis was detected with hoechst 33342 staining and Annexin V＋ PI assay.Western blotting and rhodamine 123 staining were employed to examine the changes in cellular poly（ADP-ribose）polymerase（PARP） protein expression and mitochondrial membrane potential in response to MPP＋ exposure.The effects of ruthenium red and/or MPP ＋ on calcium concentration in the cytoplasm,mitochondria and endoplasmic reticulum were evaluated using confocal microscopy.Results MPP＋ induced apoptosis and caused reduced cell viability and mitochondrial membrane potential in SH-SY5Y cells.The cells exposed to MPP ＋ showed a lowered calcium concentration in the cytoplasm and endoplasmic reticulum and an increased mitochondrial Ca2 ＋ uptake.Ruthenium red rescued MPP＋-induced apoptosis and mitochondrial membrane potential reduction,reduced PARP cleavage,and inhibited the increase of mitochondrial matrix free Ca2 ＋ in the cells exposed to MPP＋.Conclusion Mitochondrial calcium overload plays an important role in MPP＋-induced apoptosis of SH-SY5Y cells,which is closely associated with dysregulation of intracellular Ca2＋ homeostasis.

关 键 词：MPP+ 凋亡 钙稳态 神经母细胞瘤 

分 类 号：R739.4[医药卫生—肿瘤]