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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 model of human cervical cancer cell line infected with human papillomavirus type 16 (HPV-16 Psv) and investigate the anti-HPV effects of three human HDF-5 variants. METHODS: 293FT cells were co-transfected with the recombinant plasmid P16L1L2 expressing the HPV-16 pseudovirion and the Pfwb containing the green fluorescent protein (GFP) reporter gene. The virus particles were isolated and purified and then infected into the cervical cancer cell line C-33a. Meanwhile, HD-5 / N (native configuration), HD-5 / Acm (cysteine modified with Acm), or HD-5 / Abu (cysteine replaced by Abu) were each treated with 20 μg / h after fluorescence microscopy and flow cytometry virus infection rate. Results Pfwb and P16L1L2 were successfully transfected into 293FT cells and HPV-16 pseudoviruses with a titer of 2.5 × 108TU / ml were obtained. The results of microscopic and flow cytometry showed that cervical cancer cell C-33a was infected by pseudovirus and expressed GFP . The inhibitory rates of HD-5 / N, HD-5 / Acm and HD-5 / Abu on HPV-16 infected C-33a cells were 96.48 ± 5.67%, 69.02 ± 7.88% and 2.71 ± 1.53, %. The antiviral effects of HD-5 / Acm and HD-5 / Abu were significantly reduced (P <0.01) compared to HD-5 / N. Conclusion A HPV-16 pseudoviral infection model based on flow cytometry was established and used to detect antiviral activity. The spatial structure of HD-5 and cysteine in the molecule were found to play an important role in their activity against HPV infection.