人乳头瘤病毒假病毒体外感染模型的建立及人α防御素5抗病毒作用  被引量:2

Establishment of a human papillomavirus pseudovirus in vitro infection model and its application to evaluate antiviral activity of human defensin-5

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作  者:耿放[1] 王艾平[1] 陈芳[1] 申明强[1] 陈默[1] 粟永萍[1] 程天民[1] 王军平[1] 

机构地区:[1]第三军医大学军事预防医学院防原医学教研室全军复合伤研究所创伤、烧伤与复合伤国家重点实验室,重庆400038

出  处:《第三军医大学学报》2012年第6期473-476,共4页Journal of Third Military Medical University

基  金:国家自然科学基金(30771892);重庆市院士基金(CSTC2007AB5022)~~

摘  要:目的建立用人乳头瘤病毒16亚型假病毒(HPV-16 Psv)感染宫颈癌细胞模型,并研究3种不同构型人α防御素5(HD-5)抗HPV的作用。方法将表达HPV-16假病毒衣壳蛋白的重组质粒P16L1L2和含绿色荧光蛋白(GFP)报告基因的质粒Pfwb共转染293FT细胞,分离、纯化病毒颗粒后感染宫颈癌细胞株C-33a,同时分别给予20μg/ml的HD-5/N(天然构型)、HD-5/Acm(半胱氨酸被Acm修饰)或HD-5/Abu(半胱氨酸被Abu替换)处理,培养48 h后荧光显微镜观察和流式细胞分析病毒感染率。结果 Pfwb与P16L1L2成功转染293FT细胞并获得滴度达2.5×108TU/ml的HPV-16假病毒液,镜检和流式细胞术检测显示宫颈癌细胞C-33a能够被假病毒感染并表达GFP。HD-5/N、HD-5/Acm和HD-5/Abu对HPV-16感染C-33a细胞的抑制率分别为(96.48±5.67)%、(69.02±7.88)%和(2.71±1.53)%。与HD-5/N相比,HD-5/Acm和HD-5/Abu的抗病毒作用显著降低(P<0.01)。结论建立了基于流式细胞分析的HPV-16假病毒感染模型并将其用于抗病毒药物活性检测。发现HD-5的空间结构和分子中半胱氨酸对其抗HPV感染的活性有重要作用。Objective To establish a human papillomavirus16 pseudovirus(HPV16 Psv) infection model in vitro and to determine the anti-HPV effects of human α-defensin 5(HD-5) with different conformations.Methods The 293FT cells were co-transfected with HPV pseudovirus recombinant plasmid P16L1L2 and a GFP reporter plasmid Pfwb.After isolation and purification,the pseudovirus particles were used to infect cervical cancer cell line C-33a.During infection,the C-33a cells were treated respectively with 3 different configurations of HD-5,including HD-5/N with natural configuration,HD-5/Acm with Cys residues blocked by acetamidomethyl(Acm) and HD-5/Abu with Cys residues substituted by α-aminobutyric acid(Abu),at a dose of 20 μg/ml.In 48 h later,the cells were observed by fluorescent microscopy and the viral infection rate was determined by flow cytometry assay.Results The 293FT cells were successfully transfected with Pfwb and P16L1L2,and 2.5×108TU/ml of HPV-16 pseudovirus particles were finally obtained.After culturing,most of C-33a cells were infected by HPV16 pseudovirus,which displayed green fluorescence under fluorescent microscope and could be detected by flow cytometry.The inhibitory rate of HD-5/N,HD-5/Acm and HD-5/Abu on HPV infection was(96.48±5.67)%,(69.02±7.88)% and(2.71±1.53)% respectively.Compared to HD-5/N,antiviral activity of HD-5/Acm and HD-5/Abu was significantly reduced(P0.01).Conclusion A method for HPV16 pseudovirus infection and anti-HPV analysis based on flow cytometry assay is successfully established.The natural configuration of HD-5 contributes to enhance anti-HPV ability significantly,and Cys residues play important role in the antiviral activity of HD-5.

关 键 词:人乳头瘤病毒 假病毒 防御素 绿色荧光蛋白 抗病毒作用 

分 类 号:R373[医药卫生—病原生物学] R394-33[医药卫生—基础医学]

 

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