摘要
在炎症反应或在氧化应激的情况下,机体内的蛋白酶体会转变成免疫蛋白酶体,从而在炎症类疾病中发挥作用。为了研究免疫蛋白酶体在伪狂犬病毒(PRV)感染中的作用,本研究随机将25只C57BL/6小鼠分成5组,5只/组,1组对照组,4组实验组:分别为感染5 d(5 dpi)组、感染7 d(7 dpi)组、感染9 d(9 dpi)组和感染11 d(11 dpi)组。将PRV Bartha株以6.8×10^(4) TCID50的剂量经后肢皮下注射感染小鼠,每天观察其临床症状。在感染后的11 d内,小鼠出现了被毛凌乱、体重减轻和轻微的神经症状。于感染后的第5 d、7 d、9 d和11 d颈椎脱臼迫杀各组小鼠后采集脑组织,经荧光定量PCR方法检测小鼠脑组织的病毒拷贝数。结果显示,在小鼠脑组织检测出了PRV,且9 dpi组小鼠病毒的拷贝数高达4.93×10^(6)拷贝/μL;小鼠脑组织病理切片可见,小鼠产生了非化脓性脑炎,脑组织出现血管套现象并伴有炎性细胞的浸润;经荧光定量PCR方法检测各组小鼠脑组织免疫蛋白酶体亚基和促炎性细胞因子的转录水平。结果显示,各感染组小鼠脑组织免疫蛋白酶体亚基LMP2、LMP7以及LMP10的转录水平均明显上调,其中LMP2亚基的转录水平最高,与对照组相比9 dpi组小鼠脑组织LMP2亚基的转录水平上调了332倍(p<0.001);各感染组小鼠促炎性细胞因子IFN-γ、IL-6、IL-1β和TNF-α的转录水平也均上调,其中9 dpi组小鼠的IFN-γ转录水平与对照组相比上调了1420倍(p<0.01)。Western blot结果显示,各感染组小鼠脑组织免疫蛋白酶体亚基LMP2、LMP7和LMP10均被诱导表达。上述结果表明PRV Bartha株感染小鼠后,会诱发其脑组织炎症和免疫蛋白酶体的表达,且随着感染时间的延长(5 dpi~9 dpi),小鼠脑组织炎性因子与免疫蛋白酶体亚基的转录水平和蛋白表达水平均呈增加趋势。本实验为研究免疫蛋白酶体在PRV致病机理中的作用奠定了基础,并为进一步研究疱疹病毒诱发的中枢神经系统炎症反应提供了参考依据。
When in response to inflammation or oxidative stress,proteasomes in the body are converted into immune proteasomes that play a role in inflammatory diseases.To study the role of immunoproteasome in the context of pseudorabies virus(PRV)infection,25 C57BL/6 mice were randomly divided into 5 groups with 5 mice per group including 1 control group and 4 experimental groups,where mice were infected for 5(5dpi),7(7dpi),9(9dpi)and 11 days(11dpi),respectively.Mice were injected with 6.8×10^(4) TCID50 PRV Bartha strain by subcutaneous injection of the hind limb,and the development of symptoms was observed daily.The results showed that as the disease progressed,the infected mice became messy,weight loss and showed mild pruritus within 11 days.The mice were put to death by cervical vertebra dislocation and brain tissues were collected at 5,7,9,and 11 days post-infection.The quantitative analysis of the virus in mice brains was performed by using real-time PCR and the results showed that PRV was detected in experimental mice brains,the amount of virus was 4.93×10^(6) copies/μL on average in 9dpi group.Pathological changes were observed by histopathology method and the results showed that the infected mice developed nonpurulent encephalitis with a perivascular cuffing phenomenon accompanied by infiltration of inflammatory cells in brains.The mRNA transcription levels of immunoproteasome subunits LMP2,LMP7,and LMP10,and pro-inflammatory cytokines in mouse brain were quantified by real-time PCR.The results showed that LMP2,LMP7,LMP7 subunits were all up-regulated,and LMP2 was in the highest level,and its transcription level in 9dpi group was 332 times higher than that in control group(p<0.001).Proinflammatory cytokines IFN-γ,IL-6,IL-1β and TNF-α were also up-regulated in all the experimental groups.The IFN-γtranscription level of 9dpi was 1420-fold of that in control group(p<0.01).At the same time,western blot results showed that the immunoproteasome subunits LMP2,LMP7 and LMP10 were induced and expressed.The above results indicated that infection of PRV Bartha strain can drive brain inflammation and induce the expression of immunoproteasome in mouse brain.As the disease progressed(5dpi-9dpi),the transcription level and protein expression level of inflammatory factors and immunoproteasome subunits were both increased in mouse brain tissue.Our study lays the foundation for studying the function of immunoproteasome in pathogenic mechanism of PRV and provides an experimental basis for further research on the inflammatory response of central nervous system induced by herpesvirus infection.
作者
胡薇
刘强
张永莉
马泽林
肖菲
张险峰
何希君
HU Wei;LIU Qiang;ZHANG Yong-li;MA Ze-lin;XIAO Fei;ZHANG Xian-feng;HE Xi-jun(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2021年第1期65-70,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
中国农业科学院科技创新工程项目。
关键词
免疫蛋白酶体
伪狂犬病毒
脑炎
促炎性细胞因子
immunoproteasome
pseudorabies virus
encephalitis
pro-inflammatory cytokines