| 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）. |
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| 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 |
