摘要
目的:探索过表达脑红蛋白(neuroglobin,NGB)对转染了p APPswe的SH-SY5Y细胞的神经保护作用及机制。方法:成功构建过表达NGB的质粒p EGFP-NGB并转染入已预先转染了p APPswe的SH-SY5Y细胞,MTT法检测过表达NGB对该细胞存活率的影响;JC-1法检测其对细胞线粒体膜电位的影响;流式细胞术检测过表达NGB对细胞凋亡的影响;Western blotting法检测其对细胞中p-Akt、Akt和caspase-3/9表达的影响;ELISA法检测其对细胞内Aβ42生成的影响。结果:MTT结果显示,与对照组和空质粒组比较,转染NGB后,p APPswe-SH-SY5Y细胞的存活率明显提高,差异有统计学意义(P<0.05)。JC-1染色结果显示过表达NGB能够明显抑制转染p APPswe对SH-SY5Y细胞线粒体膜电位的降低作用(P<0.05)。流式细胞术结果显示过表达NGB能够抑制早、晚期细胞的凋亡。而Western blotting显示过表达NGB不仅能抑制细胞内caspase-3和caspase-9蛋白水平的表达,而且还能够促进细胞内p-Akt蛋白的表达,而这种促进作用能够被PI3K/Akt的抑制剂LY294002所抑制。ELISA结果显示过表达NGB能够明显抑制细胞内Aβ42的生成。结论:过表达NGB能够显著抑制p APPswe诱导的细胞损伤,而且还抑制与细胞凋亡密切相关的caspase-3和caspase-9等蛋白表达。NGB的神经保护作用可能是通过激活PI3K/Akt信号通路来实现的。
AIM： To study the neuroprotective roles of neuroglobin （NGB） over-expression in the SH-SYSY ceils transfected with pAPPswe. METHODS： The plasmid pEGFP-NGB was successfully constructed and transfected into the SH-SYSY cells, which were pretreated with pAPPswe. MTY assay was applied to detect the effect of NGB over-expres- sion on the cell survival rates. JC-1 staining was used to detect the level of mitochondrial transmembrane potential. The cell apoptosis was analyzed by flow cytometry. The effects of NGB over-expression on the protein level of p-Akt, Akt and caspase-3/9 were determined by Western blotting. The generation of A^42 in the cells was measured by ELISA. RE- SULTS ： The cell survival rate was remarkably increased after transfection with NGB compared with control group and emp- ty plasmid group （P 〈 0.05）. The over-expression of NGB significantly inhibited the decrease in mitochondrial membrane potential induced by pAPPswe. The over-expression of NGB inhibited the apoptosis of the cells. Furthermore, over-expres- sion of NGB not only inhibited the expression of caspase-3 and caspase-9, but also induced the production of p-Akt, which was prevented by LY294002, an inhibitor of PI3K/Akt. The generation of AI342 was inhibited in the cells with the over-ex- pression of NGB. CONCLUSION： Over-expression of NGB significantly inhibits the SH-SYSY cell injuries induced by pAPPswe and inhibits the expression of caspase-3/9, which is tightly related with cell apoptosis. Furthermore, the neuro- protective roles of NGB may be via activating PI3K/Akt signaling pathway.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2014年第12期2166-2171,共6页
Chinese Journal of Pathophysiology
基金
国家青年科学基金资助项目(No.81100948)
