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[神经球蛋白对人神经母细胞瘤细胞系氧糖剥夺及复氧诱导的自噬的影响]

[Effect of neuroglobin on oxygen-glucose deprivation and reoxygenation induced autophagy in a human neuroblastoma cell line].

作者信息

Deng S Y, Ai Y H, Gong H, Wu L, Chen C X, Wang Y M, Liu Z Y, Huang L, Peng Q Y, Zhang L N

机构信息

Department of Critical Care Medicine, Xiangya Hospital, Central South University, Changsha 410008, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2017 May 23;97(19):1505-1509. doi: 10.3760/cma.j.issn.0376-2491.2017.19.016.

Abstract

To investigate the effect of neuroglobin on oxygen-glucose deprivation and reoxygenation (OGD/R) induced autophagy in a human neuroblastoma cell line (SH-SY5Y). SH-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. Treatment 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 <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, <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. Overexpression 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.

摘要

研究神经球蛋白对人神经母细胞瘤细胞系(SH-SY5Y)中氧糖剥夺及复氧(OGD/R)诱导的自噬的影响。将质粒(或载体)转染至SH-SY5Y细胞,以建立神经球蛋白过表达(OE)的稳定细胞系。经OGD/R处理后,收集细胞用于分析mRNA(自噬相关基因5、自噬相关基因7、Beclin1和FUN14结构域包含蛋白1)水平及微管相关蛋白1轻链3的蛋白水平。此外,分离线粒体和胞质部分用于检测磷酸化依赖的自噬调节激酶1和帕金蛋白的蛋白水平。OGD/R处理显著增加了自噬相关基因5、自噬相关基因7、Beclin1和FUN14结构域包含蛋白1的mRNA水平(峰值水平分别为4.90±0.71、6.72±0.75、2.71±0.39和3.96±0.78倍,均<0.05)。处理后,帕金蛋白的蛋白水平在线粒体中增加而在细胞质中降低。与载体组相比,神经球蛋白过表达组的FUN14结构域包含蛋白1 mRNA水平显著更高(3.96±0.78对6.86±0.63倍,<0.05),而自噬相关基因5、自噬相关基因7和Beclin1的mRNA水平无显著差异。此外,神经球蛋白过表达组与载体组之间,磷酸化依赖的自噬调节激酶1或帕金蛋白的线粒体或胞质蛋白水平无显著差异。神经球蛋白过表达不影响OGD/R处理的SH-SY5Y细胞中的自噬或线粒体自噬。神经球蛋白过表达可增加FUN14结构域包含蛋白1的mRNA水平,其机制有待进一步研究。

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