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  • 徐娟,张力航,高金超,赵文娟*,殷明*.单纯疱疹病毒Ⅰ型诱导人成神经细胞瘤细胞SH-SY5Y表达β-淀粉样蛋白[J].第二军医大学学报,2018,39(10):1109-1114    [点击复制]
  • XU Juan,ZHANG Li-hang,GAO Jin-chao,ZHAO Wen-juan*,YIN Ming*.Herpes simplex virus type Ⅰ induces β-amyloid expression in human neuroblastoma cell lines SH-SY5Y[J].Acad J Sec Mil Med Univ,2018,39(10):1109-1114   [点击复制]
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单纯疱疹病毒Ⅰ型诱导人成神经细胞瘤细胞SH-SY5Y表达β-淀粉样蛋白
徐娟,张力航,高金超,赵文娟*,殷明*
0
(上海交通大学药学院神经药理学实验室, 上海 200240
*通信作者)
摘要:
目的 探讨单纯疱疹病毒Ⅰ型(HSV-1)感染对人成神经细胞瘤细胞SH-SY5Y β-淀粉样蛋白(Aβ)表达的影响。方法 通过非洲绿猴肾细胞Vero扩增HSV-1,测定病毒滴度。取感染复数(MOI)为10的3.2×108 PFU/mL HSV-1分别感染SH-SY5Y细胞12、24 h,显微镜下观察SH-SY5Y细胞的形态变化,使用PCR检测细胞中HSV-1 DNA的表达;双色免疫荧光检测SH-SY5Y细胞中Aβ和载脂蛋白E(ApoE)的表达;蛋白质印迹法检测淀粉样蛋白前体(APP)、黑素代谢酶(MME)、ApoE、糖原合成酶激酶3β(GSK-3β)、磷酸化糖原合成酶激酶3β(p-GSK-3β)蛋白的表达。结果 HSV-1感染SH-SY5Y细胞12 h后,镜下可见细胞突触减少,突起回缩;感染24 h后细胞开始聚集,呈圆形,并有细胞脱落。与磷酸盐缓冲液(PBS)对照组相比,HSV-1感染12 h后细胞中Aβ表达增加(P<0.01),ApoE蛋白表达无明显改变;感染24 h后,Aβ表达仍增加(P<0.05),但弱于12 h时(P<0.01),APOE蛋白表达增加(P<0.01)。蛋白质印迹分析结果显示,与PBS对照组比较,HSV-1感染12 h后细胞中APP蛋白的表达水平降低(P<0.05),GSK-3β总蛋白的表达水平增加(P<0.05)且磷酸化活性增强(P<0.05),MME蛋白的表达水平增加(P<0.05);感染24 h时MME蛋白的表达水平降低(P<0.05),ApoE蛋白的表达水平增加(P<0.05)。结论 HSV-1感染后可诱导SH-SY5Y细胞Aβ表达增加,在感染12 h时可能通过促进APP代谢、GSK-3β Ser9磷酸化诱导Aβ产生,而在24 h时主要是通过抑制Aβ降解促使Aβ表达增加。
关键词:  单纯疱疹病毒Ⅰ型  阿尔茨海默病  淀粉样蛋白  载脂蛋白E  淀粉样蛋白前体
DOI:10.16781/j.0258-879x.2018.10.1109
投稿时间:2018-04-04修订日期:2018-07-18
基金项目:国家自然科学基金(81471232).
Herpes simplex virus type Ⅰ induces β-amyloid expression in human neuroblastoma cell lines SH-SY5Y
XU Juan,ZHANG Li-hang,GAO Jin-chao,ZHAO Wen-juan*,YIN Ming*
(Laboratory of Neuropharmacology, School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
*Corresponding authors)
Abstract:
Objective To explore the effect of herpes simplex virus type Ⅰ (HSV-1) infection on expression of β-amyloid (Aβ) in human neuroblastoma cell lines SH-SY5Y. Methods African green monkey kidney cell lines Vero cells were used to amplified HSV-1, and the virus titers were measured. SH-SY5Y cells were infected with HSV-1 3.2×108 plaque forming unit (PFU)/mL at a multiplicity of infection (MOI) of 10 for 12 h or 24 h, and the morphological changes of the cells were observed under microscope. PCR was used to detect the expression of HSV-1 DNA. Double-color immunofluorescence assay was performed to show the expression of Aβ and apolipoprotein E (ApoE). Western blotting was used to detect the expression levels of amyloid precursor protein (APP), melanin metabolic enzyme (MME), ApoE, glycogen synthase kinase-3β (GSK-3β) and phosphorylated glycogen synthase kinase-3β (p-GSK-3β). Results After infection with HSV-1 for 12 h, the SH-SY5Y cells had synapse reduction and neurite retraction and few neurites. And after 24 h of infection, the cells began to aggregate, and were round and shed. Compared with phosphate buffer saline (PBS) control group, the expression of Aβ was significantly increased after infection with HSV-1 for 12 h (P<0.01), while the expression of ApoE protein was not significantly changed. After 24 h of infection, the expressions of Aβ and ApoE were significantly increased (P<0.05), but the expression of Aβ was significantly lower than that on 12 h of post-infection (P<0.01). Western blotting analysis showed that, compared with PBS control group, the expression of APP was significantly decreased on 12 h of post-infection (P<0.05), and the expressions of GSK-3β, p-GSK-3β and MME were significantly increased (P<0.05). However, the expression of MME was significantly decreased (P<0.05) and the expression of ApoE was significantly increased (P<0.05) 24 h post-infection. Conclusion HSV-1 infection induces the expression of Aβ in SH-SY5Y cells through promoting APP metabolism and Ser9 phosphorylation of GSK-3β on 12 h of post-infection, and inhibiting the degradation of Aβ on 24 h of post-infection.
Key words:  herpes simplex virus type Ⅰ  Alzheimer disease  amyloid  apolipoprotein E  amyloid precursor protein