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  • 陈甲,秦鲁平,赵明,陈蕾,吴涛,邓本强*.氧糖剥夺条件下p75神经营养素受体在人神经母细胞瘤细胞中的表达和作用[J].第二军医大学学报,2019,40(12):1325-1329    [点击复制]
  • CHEN Jia,QIN Lu-ping,ZHAO Ming,CHEN Lei,WU Tao,DENG Ben-qiang*.Expression of p75 neurotrophin receptor in human neuroblastoma cells under condition of oxygen-glucose deprivation and its significance[J].Acad J Sec Mil Med Univ,2019,40(12):1325-1329   [点击复制]
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氧糖剥夺条件下p75神经营养素受体在人神经母细胞瘤细胞中的表达和作用
陈甲,秦鲁平,赵明,陈蕾,吴涛,邓本强*
0
(海军军医大学(第二军医大学)长海医院脑血管病中心, 上海 200433
*通信作者)
摘要:
目的 探讨氧糖剥夺(OGD)条件下p75神经营养素受体(p75NTR)在人神经母细胞瘤细胞(SH-SY5Y)中的表达变化和作用。方法 SH-SY5Y细胞OGD模型的建立采用三气培养箱无糖无血清培养方法。模型成功建立后设立3组:无血清常规培养组(对照组)、OGD组和OGD+p75NTR竞争性阻断剂LM11A-31处理组(OGD+LM11A-31组)。在细胞培养12 h时用利用四甲基偶氮唑盐(MTT)法检测3组细胞存活率,乳酸脱氢酶(LDH)活力检测试剂盒测定LDH释放活力,流式细胞术检测凋亡细胞比例,蛋白质印迹法检测p75NTR的蛋白表达。结果 成功构建SH-SY5Y细胞OGD模型。细胞培养12 h时,对照组、OGD组和OGD+LM11A-31组细胞存活率分别为(94.80±4.06)%、(50.34±5.55)%、(64.68±4.59)%,LDH释放活力分别为(46.93±5.49)U/L、(353.09±30.67)U/L和(282.20±25.60)U/L,凋亡细胞比例分别为(1.82±0.45)%、(14.98±2.59)%和(7.36±1.98)%,p75NTR蛋白相对表达量分别为0.06±0.01、0.41±0.02和0.19±0.03,差异均有统计学意义(F=67.94、142.10、36.28、221.20,P均<0.05)。多重比较显示,OGD组细胞存活率低于对照组,LDH释放活力高于对照组,凋亡细胞比例高于对照组,p75NTR蛋白表达高于对照组,差异均有统计学意义(Bonferroni法,P'均<0.05);而LM11A-31处理后OGD+LM11A-31组细胞存活率高于OGD组,LDH释放活力低于OGD组,凋亡细胞比例低于OGD组,p75NTR蛋白表达低于OGD组,差异均有统计学意义(Bonferroni法,P'均<0.05)。结论 OGD条件下p75NTR在人神经母细胞瘤细胞SH-SY5Y中表达增加,可能促进了神经损伤和凋亡。
关键词:  神经营养素受体  氧糖剥夺  人神经母细胞瘤细胞  神经凋亡
DOI:10.16781/j.0258-879x.2019.12.1325
投稿时间:2019-07-11修订日期:2019-10-16
基金项目:上海市科委医学引导类项目(124119a8900).
Expression of p75 neurotrophin receptor in human neuroblastoma cells under condition of oxygen-glucose deprivation and its significance
CHEN Jia,QIN Lu-ping,ZHAO Ming,CHEN Lei,WU Tao,DENG Ben-qiang*
(Stroke Center, Changhai Hospital, Naval Medical University (Second Military Medical University), Shanghai 200433, China
*Corresponding author)
Abstract:
Objective To explore the expression and effect of p75 neurotrophin receptor (p75NTR) in human neuroblastoma cell line (SH-SY5Y cells) under the condition of oxygen-glucose deprivation (OGD). Methods The OGD model of SH-SY5Y cells was established by glucose-free and serum-free culturing using tri-gas incubator, and then was assigned to 3 groups, including serum-free regular culturing group (control group), OGD group and OGD+LM11A-31 (a competitive blocker of p75NTR) group. After 12 h of culturing, the cell viability was measured by methyl thiazolyl tetrazolium (MTT) assay, lactate dehydrogenase (LDH) release activity was determined by LDH activity assay kit, cell apoptosis proportion was detected by flow cytometry, and p75NTR protein expression was detected by Western blotting. Results The OGD model of SH-SY5Y cells was successfully established. Twelve hours after cell culture, the cell viability of the control, OGD and OGD+LM11A-31 groups was (94.80±4.06)%, (50.34±5.55)% and (64.68±4.59)%, the LDH release activities were (46.93±5.49) U/L, (353.09±30.67) U/L and (282.20±25.60) U/L, the proportions of apoptosis cells were (1.82±0.45)%, (14.98±2.59)% and (7.36±1.98)%, and the relative expression levels of p75NTR were 0.06±0.01, 0.41±0.02 and 0.19±0.03, respectively, and the differences were all significant (F=67.94, 142.10, 36.28 and 221.20, all P<0.05). Post hoc analysis showed that the cell viability of the OGD group was significantly lower than that of the control group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD group were significantly higher (Bonferroni test, all P'<0.05). After treatment with LM11A-31, the cell viability of the OGD+LM11A-31 group was significantly higher than that of the OGD group, and the LDH release activity, the proportion of apoptosis cells and the relative protein expression level of p75NTR of the OGD+LM11A-31 group were significantly lower (Bonferroni test, all P'<0.05). Conclusion The expression of p75NTR is increased in human neuroblastoma cell line SH-SY5Y cells under OGD condition, which may promotes neuronal injury and apoptosis.
Key words:  neurotrophin receptor  oxygen-glucose deprivation  human neuroblastoma cells  neural apoptosis