肝素酶在人β-防御素-3抗甲型流感病毒的作用研究  

Effect of heparinase on humanβ-defensin-3 against influenza A virus

作　　者：杨小余 马贵凤 唐源 罗红 江滟 YANG Xiao-yu;MA Gui-feng;TANG Yuan;LUO Hong;JIANG Yan(Department of Microbiology and Immunology,School of Clinical Laboratory,Guizhou Medical University,Guiyang 550025,China;The Laboratory Animal Center of Guizhou Medical University;Clinical Laboratory Center,Affiliated Hospital of Guizhou Medical University)

机构地区：[1]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550025 [2]贵州医科大学附属医院临床检验中心微生物免疫科 [3]贵州医科大学实验动物中心

出　　处：《中国病原生物学杂志》2022年第1期14-17,21,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No:81260249);“贵州省实验动物管理服务平台”中央引导地方科技发展专项基金项目(黔科中引地[2016]4007号)资助。

摘　　要：目的探讨肝素酶在人β-防御素-3(Humanβ-defensin 3,HBD3)抗甲型流感病毒(Influenza A virus,IAV)中的作用。方法采用CCK8法检测肝素酶对人支气管上皮细胞(BEAS-2B)的毒性情况;经肝素酶处理BEAS-2B细胞,并在4℃下与HBD3和IAV病毒液孵育2 h,分为未处理组(HBD3+IAV),肝素酶+HBD3+IAV组,提取细胞总RNA和总蛋白,采用qRT-PCR检测感染细胞中的NP mRNA表达水平,Western blot和免疫共沉淀检测HBD3和IAV HA蛋白表达水平以及HBD3和HA蛋白的相互作用。试验设未经肝素酶处理BEAS-2B细胞对照组。结果肝素酶作用48 h,其浓度在30 U/mL以下浓度时对细胞无毒性作用;HBD3干预IAV感染细胞后细胞中的NP mRNA表达水平显著降低(t=12.81、26.13、28.68,P<0.01),经肝素酶处理IAV感染的细胞其NP mRNA表达水平升高(t=-4.55,P<0.05),结果显示HBD3可抑制病毒复制,而经肝素酶处理IAV感染的细胞其病毒未减少,表明只使用肝素酶不能降低病毒感染;与未经肝素酶处理的细胞相比,经肝素酶处理的细胞中HBD3和HA蛋白表达水平降低,以及HBD3和HA蛋白的相互作用显著降低(P<0.01),表明HBD3和肝素酶共同作用可降低HA的表达来阻止IAV进入细胞。结论肝素酶和HBD3共同作用可以抑制病毒进入细胞,具有抗甲型流感病作用。Objective To investigate the effect of heparinase on humanβ-defensin-3(HBD3)against influenza A virus(IAV).Methods The toxicity of heparanase to human bronchial epithelial cells(BEAS-2 B)was detected using CCK8 test.BEAS-2 B cells were treated with heparanase,and incubated with HBD3 and IAV virus for two hours at 4℃.The experimental groups were divided into untreated group(HBD3+IAV)and heparanase+HBD3+IAV group.Total RNA and total protein were extracted.The expression of NP mRNA in infected cells was detected by qRT-PCR.The expression of HBD3 and IAV HA protein were detected by Western Blot test,and the interaction between HBD3 and HA protein was detected using immunoprecipitation.Results Heparanase at the concentration below 30 U/ml had no toxic effect on BEAS-2 B cells for 48 hours.The expression level of NP mRNA in IAV infected cells with HBD3 treatment was significantly lower than that of IAV infected cells(t=12.81,26.13,28.68,P<0.01).The expression level of NP mRNA in IAV infected cells treated with heparanase was higher than that of the HBD3+IAV group(t=-4.55,P<0.05).The results showed that HBD3 could inhibit virus replication.It indicated that heparanase could not inhibit virus infection,that heparanase did not reduce the amount of IAV.Compared with untreated cells,the expression of HBD3 and HA proteins and the interaction between HBD3 and HA proteins decreased significantly in heparanase treated cells.The results showed that the combined action of HBD3 and heparanase could reduce the expression of HA to prevent IAV entering cells.Conclusion The combined action of heparinase and HBD3 could inhibit the entry of IAV into BEAS-2 B cells and play an anti-IAV role.

关 键 词：甲型流感病毒 人β-防御素3 肝素酶 糖胺聚糖蛋白 

分 类 号：R373.1[医药卫生—病原生物学]