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  

Herpes simplex virus type Ⅰ induces β-amyloid expression in human neuroblastoma cell lines SH-SY5Y

作　　者：徐娟 张力航 高金超 赵文娟[1] 殷明[1] 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)

机构地区：[1]上海交通大学药学院神经药理学实验室,上海200240

出　　处：《第二军医大学学报》2018年第10期1109-1114,共6页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金(81471232)~~

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-SY5Yβ-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3.2×108PFU/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-3βSer9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。Objective To explore the effect of herpes simplex virus typeⅠ(HSV-1)infection on expression ofβ-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 Aβand 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 Aβwas 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 Aβand ApoE were significantly increased(P<0.05),but the expression of Aβwas 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-3βand 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 Aβin SH-SY5Y cells through promoting APP metabolism and Ser9 phosphorylation of GSK-3βon 12 h of post-infection,and inhibiting the degra

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 

分 类 号：R749.16[医药卫生—神经病学与精神病学]