人β防御素3对绿色荧光蛋白标记水疱性口炎病毒复制的作用
Effect of human β defensin 3 on green fluorescent protein labeled vesicular stomatitis virus replication
张乙进 1安利娟 2罗红 3舒莉萍 2江滟1
作者信息
- 1. 贵州医科大学附属医院临床检验中心微生物免疫科,贵州贵阳 550004;贵州医科大学医学检验学院临床微生物与免疫学教研室,贵州贵阳 550004
- 2. 贵州医科大学基础医学院免疫学教研室&细胞工程生物医药技术国家地方联合工程实验室&贵州省再生医学重点实验室,贵州贵阳 550004
- 3. 贵州医科大学实验动物中心,贵州贵阳 550025
- 折叠
摘要
目的 探讨人β防御素-3(HBD3)对绿色荧光蛋白(GFP)-水疱性口炎病毒(VSV)病毒株复制的作用.方法 取对数生长期非洲绿猴肾细胞(Vero)分为Control组、1.00感染复数(MOI)组、0.10 MOI及0.01MOI组,Control组为无病毒对照组,后3组Vero细胞用1.00 MOI、0.10 MOI、0.01 MOI VSV-GFP感染,采用噬斑形成实验观察感染第3天时各组Vero细胞存活情况,采用实时荧光定量聚合酶链式反应(RT-PCR)检测感染24 h时1.00、0.10及0.01 MOI组Vero细胞内病毒基质蛋白(M)和GFP信使RNA(mRNA)表达;取对数生长期人非小细胞肺癌细胞(A549)分为未处理组、VSV-GFP组及VSV-GFP+HBD3组,采用Imag J软件对各组镜下A549荧光进行相对定量分析,使用RT-PCR检测各组A549细胞内M和GFP mRNA表达.结果 随着病毒滴度的增加,Vero细胞内空斑形成数目增多;Vero细胞内M和GFP mRNA表达表现为0.01 MOI组＜0.10 MOI组＜1.00 MOI组(P＜0.05);VSV-GFP+HBD3组A549细胞单位面积内的荧光强度较VSV-GFP组减弱(P＜0.05),M和GFP mRNA表达较VSV-GFP组降低(P＜0.05).结论 HBD3可抑制VSV-GFP进入细胞后的病毒复制.
Abstract
Objective To investigate the effect of human β defensin-3(HBD3)on the eplication of green fluorescent protein(GFP)-vesicular stomatitis virus(VSV)strains.Methods The African green monkey kidney cells(Vero)in the logarithmic growth phase were divided into the control group,1.00 multiplicity of infection(MOI)group,0.10 MOI group,and 0.01 MOI group.The control group was the virus-free control group,and the other three groups Vero cells were infected with 1.00 MOI,0.10 MOI and 0.01 MOI VSV-GFP.The survival of Vero cells in each group was observed by plaque formation assay for 3 days of infection,and the mRNA expressions of viral matrix protein(M)and GFP in Vero cells infected in 1.00,0.10 and 0.01 MOI groups in the 24 hour were detected by real-time quantitative polymerase chain reaction(RT-PCR).Human non-small cell lung cancer cells(A549)in the logarithmic growth phase were divided into the untreated group,VSV-GFP group and VSV-GFP+HBD3 group.The VSV-GFP group and VSV-GFP+HBD3 group were infected with 0.10 MOI VSV-GFP for 24 h,the latter was added with 500 ng/L HBD3 for 2 h of infection,and the relative quantitative analysis of the fluorescence under the microscope of each group was carried out by IMG J software.RT-PCR was used to detect the expression of M and GFP mRNA in A549 cells.Results With the increase of viral titer,the number of plaques formed in Vero cells increased.The mRNA expression of M and GFP in Vero cells showed 0.01 MOI group＜0.10 MOI group＜1.00 MOI group(P＜0.05),the fluorescence intensity per unit area of A549 cells in VSV-GFP+HBD3 group was lower than that in VSV-GFP group(P＜0.05),and the mRNA expression of M and GFP was lower than that in VSV-GFP group(P＜0.05).Conclusion HBD3 can inhibit viral replication after VSV-GFP enters cells.
关键词
水疱性口炎/病毒/感染/病毒复制/抗菌肽/人β防御素-3/抗病毒Key words
vesicular stomatitis/virus/infection/virus replication/antimicrobial peptides/human beta defensin-3/antiviral引用本文复制引用
基金项目
贵州省科技计划项目(黔科合平台人才[2019]5610)
出版年
2024
NETL
NSTL
万方数据