雄黄纳米颗粒在基因水平上抑制腺病毒复制的实验研究

Study on realgar nanoparticles inhibition of adenovirus replication at the gene level

目的建立人腺病毒3型（HAdV-3）感染Hep-2细胞模型，采用荧光定量PCR（Real-timePCR）技术，观察雄黄纳米微粒在基因水平上对HAdV-3DNA表达的影响。方法将实验分为4组，即Hep-2细胞对照组、HAdV-3病毒对照组、雄黄纳米微粒组和利巴韦林组。在HAdV．3感染Hep．2细胞后，分别加入雄黄纳米微粒和利巴韦林作用24h，提取HAdV-3感染Hep-2细胞的总DNA，建立Real-timePCR反应体系，测定各实验组HAdV-3的病毒载量。结果Hep-2细胞对照组无扩增曲线，Ct〉35，腺病毒病原体检测阴性；雄黄纳米微粒组、利巴韦林组和HAdV-3病毒对照组扩增曲线ct分别为29．30±0．08、33．05±1．29、26．01±0．25，腺病毒病原体检测阳性。与HAdV-3病毒对照组病毒复制量（66699932±23．85）相比，利巴韦林组病毒复制量（459124．84±12．82）明显降低，差异有统计学意义（P〈0．05）；雄黄纳米微粒组病毒复制量（912435．44±16．57）较HAdV-3病毒对照组病毒复制量有所降低（P〈0．05）。结论建立的HAdV-3感染Hep．2细胞模型可靠，荧光定量PCR方法灵敏性高、特异性强，雄黄纳米微粒对腺病毒的核酸复制有-定的抑制作用。

Objective Modeling HAdV-3 infect Hep-2 cells in vitro. The effect of realgar nanoparticles on the expression of HAdV-3 is detected by using fluorescent quantitative PCR. Method The experiment is divided into four groups： Hep-2 cells control group, HAdV-3 virus control group, realgar nanoparticle group and ribavirin group. In order to detect HAdV-3 viral load, add realgar nanoparticles and ribavirin in vitro and remain that vitro for 24 hours when HAdV-3 has infected Hep-2 cells, extract total DNA of Hep-2 cells infected by HAdV-3, and establish Real-time PCR reaction system of every experimental groups. Result The Hep-2 cells group has no amplification curve, the Ct value is greater than 35,which illustrate HAdV-3 pathogen detection is negative. However, realgar nanoparticles group, ribavirin group and the HAdV-3 group have amplification curve, the Ct values are 29.30 ± 0. 08, 33.05± 1.29, 26.01 ± 0. 25 respectively, which illustrate HAdV-3 pathogen detection is positive. The viral copy amount of the adenovirus group（66 699 932±23.85） is more than that of realgar nanopartieles group （912 435.44 ± 16. 57）, and much greater than that of ribavirin group （459 124.84 ± 12.82） （ P 〈 0. 05 ）. Conclusion The model of Hep-2 cell infected by HAdV-3 is reliable. The method of quantitative PCR is sensitive and specific. Realgar nanoparticles have a certain inhibition role for adenovirus nucleic acid replication.