单纯疱疹病毒Ⅱ型潜伏、激活细胞模型建立

Establishment of a cell model system of herpes simplex virus typeⅡ latent infection and reactivation in SH-SY5Y cells

摘要:

摘要【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20，40，60，80，100，120，140 μmol/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、100 MOI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0.5 h、1.0 h、1.5 h、2.0 h、2.5 h，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、125 μmol/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】60 μmol/L ACV的存在最适合HSV-2在SH-SY5Y细胞中建立潜伏状态；1-10 MOI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14 d；43℃，1.5 h及75 μmol/L Forskolin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24 h到72 h，细胞变性、坏死的程度、数量随感染时间延长而增加；HSV-2 LAT、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。

Abstract:

Abstract: [Objective] To establish an experimental culture system of herpes simplex virus typeⅡ(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 inoculated 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 125 μmoL/L to activate the virus from latency. PCR was used to authenticate HSV-2 latent infection and reactivation. The morphological 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 infection 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.