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.DOC

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( 耿 放，王艾平，陈 芳，申明强，陈 默，粟永萍，程天民，王军平　（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） [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，并研究三种不同构型人α防御素5（HD-5）的抗HPV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（P0.01）。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的空间结构和分子间半胱氨酸对其抗HPV感染的活性有重要作用。 [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 [中图法分类号] [文献标志码] A Establishment of a human papillomavirus pseudovirus infection model and its application for analysis of antiviral ability of human defensin-5 Geng Fang, Wang Aiping, Chen Fang, Shen Mingqiang, Chen Mo, Su Yongping, Chen Tianmin, Wang Junping（State Key Lab of Trauma, Burns Combined Injury, Institute of Combined Injury of PLA, Third Military Medical University, Chongqing 400038, China.） ［Abstract］ Objective To establish a human papillomavirus16 pseudovirus (HPV16 Psv) infection model in vitro for the study of 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 three 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 20ug/ml. Fourty-eight hours later, the cells were observated by fluorescent microscope and the v