细胞因子/趋化因子在单纯疱疹病毒脑炎小鼠表达及生物信息学分析

Expression profiles of cytokines/chemokines and bioinformatic analysis of the proteins with differential expression in the mice with herpes simplex virus encephalitis

目的 探讨单纯疱疹病毒脑炎(herpes simplex encephalitis, HSE)中细胞因子/趋化因子谱的表达情况,确定差异表达的因子及其相关的生物信息学作用。方法 单纯疱疹病毒1型(HSV-1)接种于Vero细胞,Reed-Muench法测定病毒滴度;实验组小鼠鼻腔接种HSV-1,建立动物感染模型,观察至接种后第7天,处死小鼠,左半脑制备石蜡切片用于病理学检测,右半脑制备脑组织匀浆用于观察其致Vero细胞病变作用及高通量细胞因子/趋化因子表达谱芯片检测。GO、KEGG和PPI等分析技术用于差异表达蛋白的生物信息学研究。结果 实验组小鼠接种HSV-1后几乎都出现了病毒性脑炎的症状(发病率为100%),7 d内15只小鼠中死亡3只(死亡率20%),而对照组小鼠均正常,但实验组动物的发病率和死亡率与正常对照组相比差异无统计学意义(P>0.05)。脑组织病理学检测发现实验组小鼠脑组织轻度水肿伴大量炎性细胞浸润,见少量出血灶,对照组小鼠脑组织正常。实验组小鼠脑组织匀浆接种Vero细胞后发现大量细胞脱落、融合,出现明显的细胞病变效应,对照组细胞无明显改变。脑组织匀浆经PCR扩增出了病毒UL36的基因片段,而对照组未扩增出。细胞因子/趋化因子芯片于实验组筛选出了7个差异表达蛋白,GO富集分析发现其涉及多个生物学过程,与炎性细胞的活化和趋化有关;KEGG通路富集分析筛选出了7个有意义的信号通路,涉及TNF信号通路和NOD样受体信号通路等;PPI分析发现大多数差异表达蛋白组成了一个相互作用网络,其中IL-6、IL-1、IFN-γ和TNF-α是该网络的主要因子。结论 单纯疱疹病毒脑炎小鼠模型中筛选出了7种差异表达的细胞因子/趋化因子,和与之相关的信号通路可能参与了HSE的病理生理机制。

Objective To explore the differently expressed cytokines and chemokines and analyze the associated bioinformatic characteristics of the selected factors to understand the pathways that lead to herpes simplex encephalitis (HSE).MethodsVero cells were used for the titer determination of herpes simplex virus type 1 (HSV-1) by Reed-Muench method.Mice in the experimental group were inoculated intranasally with HSV-1 to establish the virus infected animal models and observed to the 7thday after inoculation.The mice were killed and the brain tissues were removed to prepare the paraffin sections with left cerebral hemisphere for pathological examination and brain tissue homogenate with right cerebral hemisphere for the cytopathic effects and cytokine chip assay.GO,KEGG,and PPIs analyses were employed to investigate the biological process (BP),pathways and interaction network of the differently expressed proteins (DEPs) in HSE mice.ResultsAll of the animals suffered from HSE (100%) and three out of 15 mice died (20%) in the experimental group.However,all of the mice in control group were normal.Interestingly,there were no significant differences of the mortality and morbidity between the experimental group and the control (P>0.05).Virus-infected brain tissues showed inflammatory damages such as edema,hemorrhagic lesions,and immune cell infiltration.Conversely,no lesions were found in the control.A large number Vero cells were abscission and fusion after inoculated by the brain homogenate separated from the experimental group mice,no changes were found in control group.The virus gene of UL36 was detected by PCR only in the homogenate in the experimental mice brain.Seven DEPs and various proteins-related signal pathways were identified in HSE mice.Multiple biological processes which mainly implicated in immune cell activation and chemotaxis were screened by GO analysis;seven signal paths were selected by KEGG,such as TNF signal pathway and NOD signal pathway,et al.The DEPs constituted a pivotal protein interaction network by PPI,and IL-6,IL-1,IFN-γ and TNF-α were the main factors in the network.Conclusion Seven DEPs and a series of related signal pathways are selected,which might associate with the pathophysiological mechanisms responsible for HSE.