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单纯疱疹病毒2型潜伏感染激活模型的建立及潜伏相关转录子开放读码框架抑制后对潜伏相关转录子基因表达的影响

Establishment of re-activated latent herpes simplex virus 2 infection model and inhibitory effect of latencyasscciated transcript open reading frame on expression of latency-asscciated transcript gene

摘要:

目的 研究单纯疱疹病毒2型(HSV-2)潜伏感染激活中潜伏相关转录子(LAT)开放读码框架(ORF)抑制后对LAT基因表达的影响。方法 建立HSV-2潜伏感染复发的神经细胞(SHSY5Y)模型。运用相差显微镜观察HSV-2潜伏及激活后SH-SY5Y细胞形态的变化。对病毒在细胞中的潜伏及激发进行PCR验证并测序。设计3对siRNA,激活诱导后转染SH-SYSY细胞,相差显微镜观察细胞形态变化,RT-PCR半定量检测转染前后LAT ORF表达的改变。结果在存在60 μmol/L阿昔洛韦(ACV)的环境,HSV-2在SH-SY5Y细胞中建立潜伏状态。病毒在SH-SY5Y细胞中最长可潜伏14d。43℃ 1.5 h诱导病毒潜伏激发,而相差显微镜观察发现,病毒激发后细胞病变从24h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、糖蛋白G(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。LAT ORF-siRNA转染细胞后,LAT ORF mRNA的表达水平在转染后24、36和48 h分别减少了39%、51%和60%。结论 抑制LAT ORF后能够抑制HSV-2 LAT基因的表达,为进一步研究LAT ORF在病毒潜伏感染复发中的作用提供了依据。

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abstracts:

Objective To investigate inhibitory effect of targeting herpes simplex virus (HSV)-2 latency-asscciated transcript (LAT) open reading frame(ORF) on expression of LAT gene in activated latent HSV-2 infection. Methods The neural cell model of re-activated latent HSV-2 infection was established using the cell line SH-SY5Y. The morphology of SH-SY5Y cells with latent and re-activated HSV-2 infection was studied with phase-contrast microscopy, and PCR was used to verify the latent and re-activated HSV-2 infection. Three pairs of siRNA targeting HSV LAT mRNAwere designed and transfected into SH-SY5Y cells using LipofectamineTM 2000 reagent. The change in SH-SY5Y morphology under phase-contrast microscope was further studied. Moreover, the expression levels of HSV LAT mRNA before and at different time points after transfection were assayed by RT-PCR. Results Established latent HSV-2 infection in SH-SY5Y cells was completed in the presence of 60 μmol/L Aciclovir(ACV). Observational study showed that the longest latency of HSV-2 infection in SH-SY5Y cells was 14 days. After induced viral re-activation with heat stress at 43°C for 1.5 h, phase-contrast microscopy revealed a range of cytopathic effects in SH-SY5Y between 24 h and 72 h, with increasing cell degeneration and necrosis over time. PCR amplification of HSV-2 LAT genes and electrophoresis of PCR products confirmed the latent and re-activated infection of virus in the cells. There was a reduction in HSV LAT mRNA expression in the cells transfected with HSV LAT ORF-siRNA by 39%,51% and 60% at 24, 36 and 48 h, respectively. Conclusion Inhibition of LAT ORF may lead to down-regulated expression of HSV-2 LAT and glycoprotein G (gG) genes, which provides robust evidence for further studies on the role of LAT ORF in re-activation of latent HSV-2 infection.

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作者: 孙朝晖 [1] 徐少珊 [1] 危敏 [2] 杨慧兰 [3]
分类号: R318
栏目名称: 论著
DOI: 10.3760/cma.j.issn.1674-1927.2011.04.006
发布时间: 2011-11-24
基金项目:
广东省自然科学基金 全军医学科学技术研究“十一五”计划 中国博士后科学基金
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