摘要
目的探讨脑红蛋白(Ngb)对氧糖剥夺/复氧复糖(OGD/R)诱导的人神经母细胞瘤细胞系(SH-SY5Y)自噬的影响。方法通过在SH-SY5Y细胞中转染Ngb质粒,建立Ngb过表达(NGB OE)及相应的空载(Vector)稳转株。细胞行OGD/R处理。聚合酶链反应(PCR)检测自噬相关基因(Atg5、Atg7、BECN1)及FUN14结构域包含蛋白-1(FUNDC1) mRNA表达水平,免疫印迹方法检测自噬相关基因(LC3 Ⅱ/Ⅰ)蛋白表达水平。差速离心法分离线粒体及细胞质,分别使用免疫印迹方法检测帕金森病相关蛋白(Parkin、Pink1)表达水平。结果OGD/R处理后,自噬相关基因Atg5、Atg7、BECN1、FUNDC1 mRNA水平升高(最高分别为4.90±0.71、6.72 ±0.75、2.71±0.39及3.96±0.78倍,均P〈0.05),细胞质中Parkin减少而线粒体中Parkin显著增加。过表达Ngb不能改变自噬相关基因Atg5、Atg7、BECN1 mRNA水平,细胞质或线粒体中Parkin无显著变化。但可以增加FUNDC1 mRNA含量(3.96±0.78倍比6.86±0.63倍,P〈0.05)。结论Ngb不能影响OGD/R诱导的SH-SY5Y细胞自噬或线粒体自噬水平,而对FUNDC1基因的表达有促进作用,其作用机制需要进一步深入研究。
ObjectiveTo investigate the effect of neuroglobin on oxygen-glucose deprivation and reoxygenation (OGD/R) induced autophagy in a human neuroblastoma cell line (SH-SY5Y).MethodsSH-SY5Y cells were transfected with plasmids (or vector) to establish a stable cell line of NGB overexpression (OE). After treated with OGD/R, cells were collected for the analyses of mRNA (Atg5, Atg7, BECN1 and FUNDC1) and protein levels of LC3. Furthermore, mitochondrial and cytosolic fractions were isolated for protein levels of PINK1 and Parkin.ResultsTreatment of OGD/R significantly increased the levels of mRNA of Atg5, Atg7, BECN1 and FUNDC1 (peak levels were 4.90±0.71, 6.72±0.75, 2.71±0.39 and 3.96±0.78 fold, all P〈0.05). The protein level of Parkin increased in mitochondria and decreased in cytoplasm after the treatment. Compared with the vector group, Ngb OE group showed a significant higher level of FUNDC1 mRNA (3.96±0.78 versus 6.86±0.63 fold, P〈0.05), while Atg5, Atg7 and BECN1 mRNA levels showed no significant difference. Moreover, the mitochondrial or cytosolic protein levels of PINK1 or Parkin showed no significant difference between Ngb OE and vector group.ConclusionsOverexpression of Ngb can not affect autophagy or mitohpagy in OGD/R treated SH-SY5Y cells. Overexpression of Ngb can increase the mRNA level of FUNDC1 and the mechanism needs further study.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2017年第19期1505-1509,共5页
National Medical Journal of China
基金
国家自然科学基金(81401099,81671960)
关键词
脓毒症
缺氧
脑
线粒体
氧糖剥夺
Sepsis
Hypoxia,brain
Mitochondria
Oxygen glucose deprivation