单纯疱疹病毒Ⅱ型潜伏、激活细胞模型建立

【目的】建立单纯疱疹病毒Ⅱ型(HSV-2)潜伏感染人神经母细胞瘤细胞株SH-SY5Y及激活的细胞模型。【方法】分别加入20,40,60,80,100,120,140μmol/L的阿昔洛韦(ACV),观察对SH-SY5Y细胞生物性状的影响;在ACV存在的情况下,分别将含有0.1、1、10、100MOI的病毒液接种SH-SY5Y细胞,运用相差显微镜观察病毒对细胞的影响,确定潜伏的建立;分别用41℃、42℃、43℃、44℃、45℃加热0.5h、1.0h、1.5h、2.0h、2.5h,观察加热时间及温度诱导HSV-2在SH-SY5Y细胞中激发的最适条件;加入25、50、75、100、125μmol/L福斯高林(Forskolin)诱导病毒在细胞中激活,探讨诱导的最佳浓度;对HSV-2在SH-SY5Y细胞中的潜伏及激发进行验证并测序;运用相差显微镜观察病毒激活后细胞形态的变化。【结果】60μmol/LACV的存在最适合HSV-2在SH-SY5Y细胞中建立潜伏状态;1-10MOI的感染量均能取得较好的病毒潜伏及激发效果;通过观察,病毒在SH-SY5Y细胞中最长可潜伏14d;43℃,1.5h及75μmol/LForskolin均为诱导病毒潜伏激发的最佳条件;相差显微镜观察病毒激发后细胞病变,从24h到72h,细胞变性、坏死的程度、数量随感染时间延长而增加;HSV-2LAT、gG基因PCR扩增及电泳结果,证实病毒在细胞中的潜伏及激活。【结论】初步在人神经母细胞瘤细胞株SH-SY5Y上建立了HSV-2潜伏感染及激活的细胞模型,为下一步研究HSV-2的潜伏与激发机理,了解HSV-2的致病机制打下基础。

Establishment of a cell model system of herpes simplex virus type Ⅱ latent infection and reactivation in SHSY5Y cells

Objective]To establish an experimental culture system of herpes simplex virus type Ⅱ(HSV-2)latent infection and reactivation in SH-SY5Y cells.Methods]Changes of biological character were observed after 20,40,60,80,100,120 and 140 μmoL/L ACV were added into cell cultures,and also the morphological observation was detected with phase-contrast microscopy after HSV-2 was inoeulated into SH-SY5Y cells using MOI of 0.1,1,10 and 100.The optimum condition of time and temperature was approached using temperature of 41℃,42℃,43℃,44℃ and 45℃,and time of 0.5 h,1.0 h,1.5 h,2.0 h and 2.5 h to induce HSV-2 reactivation.The optimum concentration of Forskolin was also decided using 25,50,75,100 and 1 25 μmoL/L to activate the virus from latency.PCR was used to authenticate HSV-2 latent infection and reactivation.The morchological changes were observed when the virus was reactivated from latency.Results ] The optimum concentration of ACV was 60 μmoL/L to establish latency in SH-SY5Y cells.Suitable infective dose of HSV-2 was 1-10 MOI to construct latency and reactivation in SH-SY5Y cells.The time of virus latency in SH-SY5Y cells could reach up to 14 d.Heat stress of 43℃,1.5 h and 75 μmoL/L Forskolin were the optimum condition to induce virus reactivation.The morphologic changes in SH-SY5Y cells recurred by HSV-2:Cytopathic effects were more and more obvious with time lasting from 24 h to 72 h after reactivation from latency.PCR and results of electrophoresis proved the cell model of latent infeorion and reactivation was set up successfully.Conclusions]The cell model system of HSV-2 latent infection and reactivation in SH-SY5Y cells was established.The research provided usefulness for study on HSV-2 of latency.reactivation and pathogenic mechanism.