胞质和核定位α-突触核蛋白过表达对人神经母细胞瘤细胞的影响

Effects of cytoplasm localized or nuclear localized overexpression α-synuclein on SH-SY5Y cells

探讨α-突触核蛋白在人神经母细胞瘤(SH-SY5Y)细胞中定位过表达的影响.研究应用PLVU-SNCA-NES(NLS)-2A-EGFP重组慢病毒载体感染SH-SY5Y细胞,通过观察绿色荧光蛋白(EG-FP)检测目的基因在细胞中的表达情况.形态观察与MTT检测相结合,分析胞质和核定位α-突触核蛋白过表达对SH-SY5Y细胞增殖的影响,同时测定鱼藤酮、H2O2对胞质和核定位α-突触核蛋白过表达SH-SY5Y细胞活性的影响.MitoTracker染色法测定胞质和核定位α-突触核蛋白过表达的SH-SY5Y细胞中线粒体数目的变化.结果发现,α-突触核蛋白胞质定位过表达使得SH-SY5Y细胞培养96h时活性显著增强,24h、48h、96h时细胞中线粒体的数目显著增加,增长比率分别为91.18%、41.15%、50.21%;α-突触核蛋白核定位过表达的SH-SY5Y细胞相对正常细胞表现出较低的细胞活性,在培养48h、72h后细胞存活率显著下降.400nmol/L鱼藤酮损伤24h后,α突触核蛋白核定位过表达的SH-SY5Y细胞活性显著高于正常细胞鱼藤酮损伤组.50～800μmol/LH2O2处理条件下,α-突触核蛋白的定位过表达对SH-SY5Y细胞活性的影响并不显著.研究结果表明α-突触核蛋白胞质定位过表达表现出一定的神经保护作用,α-突触核蛋白核定位过表达也能使SH-SY5Y细胞的活性发生改变.

To study the effects of overexpression α-synuclein localized in cytoplasm or in nuclear on viability of SH-SY5Y cells. The recombinant plasmid PLVU＂ SNCA NES（NLS） 2A-EGFP was transfected into SH-SY5Y cells by lentivirus vector. Then the expression of reporter gene EGFP is detected by fluorescence microscopy; MTT to analyze the viability of SH-SY5Y, SH SY5Y（NES）and SH-SYSY（NLS）cells;MitoTraker Red to detect the amount of mitochondria. After transfected with the recombinant plasmid PLVU-SNCA NES（NLS）-2A EGFP by lentivirus vector for 48 h, the expression of EGFP is detected. Both the stable genetic SH-SY5Y（NES） and SH SY5Y（NLS）are established. The viability of SH SY5Y（NES）cells is significantly enhanced compared with normal control at 96 h, and the amount of mitochondria in SH-SY5Y（NES）is significantly increased at 24 h,48 h and 96 h, and the increasing rate is 91. 18 %, 41. 15 % and 50.21%, respectively. While the viability of SH-SYSY （NLS）is decreased following the transfection, it is surprising to find that following the administration of 400 nmol/L rotenone, the viability of SH-SY5Y（NLS）is much higher than SH-SY5Y cells. But with the treatment of 50-800μmol/L H2O2 , there is no significant difference in survival rate occurred among all kinds of cells. In summary,α-synulein cytoplasm overexpression may enhance the viability of SH-SY5Y cells. α-synuelein nuclear localization overexpression can alsochange the viability of SH-SY5Y cells.