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【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。
【Objective】 To establish a cell model of SH-SY5Y and its activation in latent infection of human herpes simplex virus type 2 (HSV-2). 【Method】 Acyclovir (ACV) of 20, 40, 60, 80, 100, 120 and 140μmol / L were respectively added to observe the effect on the biological properties of SH-SY5Y cells. In the presence of ACV, The cells were infected with 100MOI of virus solution, and the cells were infected with 41 ℃, 42 ℃, 43 ℃, 44 ℃ and 45 ℃ for 0.5h, 1.0h, 1.5h h, 2.0h and 2.5h respectively. The optimal conditions for the induction of HSV-2 in SH-SY5Y cells were observed by heating time and temperature. After induced by Forskolin at 25, 50, 75, 100 and 125μmol / L, The activation of HSV-2 in SH-SY5Y cells was verified and sequenced. The morphological changes of HSV-2 cells after activation were observed by phase contrast microscopy. 【Result】 The results showed that 60μmol / LACV was most suitable for establishing latent state of HSV-2 in SH-SY5Y cells. The infection rate of 1-10MOI could achieve better virus latency and provoking effect. The longest incubation period of 14d was 43 ℃, 1.5h and 75μmol / LForskolin were the best conditions for inducing latent incubation of virus. The number of cell degeneration and necrosis was observed with phase contrast microscopy after 24 h to 72 h Infection time prolonged and increased; HSV-2LAT, gG gene PCR amplification and electrophoresis results, confirmed the virus in the cell latency and activation. 【Conclusion】 The cell model of latent infection and activation of HSV-2 in human neuroblastoma cell line SH-SY5Y was initially established. To investigate the latent and challenge mechanism of HSV-2 and to understand the pathogenesis of HSV-2 lay the foundation.