水痘-带状疱疹病毒对小鼠脑星形胶质细胞内NLRP3炎症小体的作用  

Effect of varicella-zoster virus on nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome in mouse brain astrocytes

作　　者：冯虹燕 王兴旺 赵阳 杨慧兰 Feng Hongyan;Wang Xingwang;Zhao Yang;Yang Huilan(Guangzhou University of Chinese Medicine,Guangzhou 510006,China;Department of Dermatology,General Hospital of Southern Theater Command of PLA,Guangzhou 510010,China)

机构地区：[1]广州中医药大学,广州510006 [2]解放军南部战区总医院皮肤科,广州510010

出　　处：《中华皮肤科杂志》2024年第6期524-529,共6页Chinese Journal of Dermatology

基　　金：国家自然科学基金(81673072);南部战区总医院科技计划课题(2021NZC015)。

摘　　要：目的研究水痘-带状疱疹病毒(VZV)感染小鼠脑星形胶质细胞后,不同VZV浓度及感染时间对细胞内核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)炎症小体相关蛋白的影响。方法用不同稀释度[100(原倍未稀释)~10-7]的VZV无细胞悬液感染小鼠脑星形胶质细胞,光学显微镜下观察各组细胞病变效应,以确定后续实验中VZV浓度和时间。用100、10-2、10-4、10-6稀释度的VZV无细胞悬液分别感染小鼠脑星形胶质细胞,于第5天收样;用1.0×105/ml(半数组织感染量)的VZV无细胞悬液感染小鼠脑星形胶质细胞,分别在感染第3、5、7、9天收样;均以正常培养的小鼠脑星形胶质细胞为对照组。用Western印迹法检测各组中VZV糖蛋白E(gE)、NLRP3、胱天蛋白酶1(caspase-1)和白细胞介素1β(IL-1β)的蛋白表达水平,比较不同VZV浓度及感染时间对小鼠脑星形胶质细胞内蛋白表达的影响。符合正态分布的数据比较采用单因素方差分析,两两多重比较采用LSD-t检验。结果100稀释度VZV感染细胞,第3天可观察到细胞数目减少,部分细胞胞体形状变得不规则,失去原有形态,细胞质呈颗粒样,细胞核结构不清,部分细胞融合,形成“多核巨细胞”;第5天,100稀释度组≥50%细胞出现上述改变,部分细胞间隙增大成网状;第9天,100稀释度组至10-6稀释度组细胞间网状间隙扩大,≥50%细胞出现明显的细胞病变表现;第10天,对照组可见部分细胞出现胞质空泡化、脱落漂浮。据此设置实验浓度梯度范围为100~10-6稀释度,观察时间为第3天至第9天。Western印迹结果显示,VZV感染组中VZV gE、NLRP3、caspase-1、IL-1β的蛋白表达量均高于对照组,且不同VZV浓度组间(F=21.45、43.82、21.85、29.53,均P<0.05)、不同感染时间组间(F=224.10、78.79、186.50、85.08,均P<0.05)蛋白表达量差异有统计学意义。随着稀释倍数提高,炎症小体相关蛋白(NLRP3、caspase-1、IL-1β)表达量降低,100稀释度�Objective To investigate the effect of different concentrations and infection durations of varicella-zoster virus(VZV)on nucleotide-binding and oligomerization domain-like receptor protein 3(NLRP3)inflammasome-related proteins in mouse brain astrocytes.Methods Mouse brain astrocytes were infected with different dilutions(100[undiluted original concentration]-10-7)of cell-free VZV suspension,and the cytopathic effect was observed under a microscope in each group to determine the concentration and infection duration of VZV for subsequent experiments.Some mouse brain astrocytes were infected with cell-free VZV suspension at dilutions of 100,10-2,10-4,and 10-6,and samples were collected on day 5;some other mouse brain astrocytes were infected with cell-free VZV suspension at the median tissue culture infective dose(TCID50)of 1.0×105/ml,and the samples were collected on days 3,5,7,and 9,separately;normally cultured mouse brain astrocytes served as control groups in the above experiments.Western blot analysis was performed to determine the protein expression of VZV glycoprotein E(gE),NLRP3,caspase-1,and interleukin-1β(IL-1β)in mouse brain astrocytes in each group.One-way analysis of variance was used for comparisons of data with a normal distribution,and least significant difference-t test was used for multiple comparisons.Results On day 3 after infection with VZV at the dilution of 100,the number of cells decreased,some cells became irregular,lost their original shapes,and showed granular cytoplasm and an unclear nucleus structure,while some cells fused and formed multinucleated giant cells;on day 5,≥50%of cells showed the aforementioned changes and some intercellular spaces were dilated giving a network-like appearance in the 100-dilution group;on day 9,the 100--10-6-dilution groups all showed dilated intercellular spaces arranged in networks and cytopathic effect in≥50%of cells;on day 10,some cells in the control group exhibited cytoplasmic vacuolation,exfoliation and flotation.Based on these results,the foll

关 键 词：疱疹病毒3型 人 星形胶质细胞 Nod样受体蛋白3 半胱氨酸天冬氨酸蛋白酶1 白细胞介素1Β 

分 类 号：R751[医药卫生—皮肤病学与性病学]