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。
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.
出处
《中华生物医学工程杂志》
CAS
2011年第4期319-324,共6页
Chinese Journal of Biomedical Engineering
基金
广东省自然科学基金（9151001002000001）
全军医学科学技术研究＂十一五＂计划（06J008）
中国博士后科学基金（20070410831）
