中华生物医学工程杂志
中華生物醫學工程雜誌
중화생물의학공정잡지
CHINESE JOURNAL OF BIOMEDICAL ENGINEERING
2011年
4期
319-324
,共6页
孙朝晖%徐少珊%危敏%杨慧兰
孫朝暉%徐少珊%危敏%楊慧蘭
손조휘%서소산%위민%양혜란
开放读码框架%疱疹病毒2型%RNA干扰%潜伏相关转录子%SH-SY5Y细胞
開放讀碼框架%皰疹病毒2型%RNA榦擾%潛伏相關轉錄子%SH-SY5Y細胞
개방독마광가%포진병독2형%RNA간우%잠복상관전록자%SH-SY5Y세포
Open reading frames%Herpesvirus 2%RNA interference%Latency-asscciated transcript%SH-SY5Y cells
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。
目的 研究單純皰疹病毒2型(HSV-2)潛伏感染激活中潛伏相關轉錄子(LAT)開放讀碼框架(ORF)抑製後對LAT基因錶達的影響。方法 建立HSV-2潛伏感染複髮的神經細胞(SHSY5Y)模型。運用相差顯微鏡觀察HSV-2潛伏及激活後SH-SY5Y細胞形態的變化。對病毒在細胞中的潛伏及激髮進行PCR驗證併測序。設計3對siRNA，激活誘導後轉染SH-SYSY細胞，相差顯微鏡觀察細胞形態變化，RT-PCR半定量檢測轉染前後LAT ORF錶達的改變。結果在存在60 μmol/L阿昔洛韋(ACV)的環境，HSV-2在SH-SY5Y細胞中建立潛伏狀態。病毒在SH-SY5Y細胞中最長可潛伏14d。43℃ 1.5 h誘導病毒潛伏激髮，而相差顯微鏡觀察髮現，病毒激髮後細胞病變從24h到72 h，細胞變性、壞死的程度、數量隨感染時間延長而增加。HSV-2 LAT、糖蛋白G(gG)基因PCR擴增及電泳結果證實病毒在細胞中的潛伏及激活。LAT ORF-siRNA轉染細胞後，LAT ORF mRNA的錶達水平在轉染後24、36和48 h分彆減少瞭39％、51％和60％。結論 抑製LAT ORF後能夠抑製HSV-2 LAT基因的錶達，為進一步研究LAT ORF在病毒潛伏感染複髮中的作用提供瞭依據。
목적 연구단순포진병독2형(HSV-2)잠복감염격활중잠복상관전록자(LAT)개방독마광가(ORF)억제후대LAT기인표체적영향。방법 건립HSV-2잠복감염복발적신경세포(SHSY5Y)모형。운용상차현미경관찰HSV-2잠복급격활후SH-SY5Y세포형태적변화。대병독재세포중적잠복급격발진행PCR험증병측서。설계3대siRNA，격활유도후전염SH-SYSY세포，상차현미경관찰세포형태변화，RT-PCR반정량검측전염전후LAT ORF표체적개변。결과재존재60 μmol/L아석락위(ACV)적배경，HSV-2재SH-SY5Y세포중건립잠복상태。병독재SH-SY5Y세포중최장가잠복14d。43℃ 1.5 h유도병독잠복격발，이상차현미경관찰발현，병독격발후세포병변종24h도72 h，세포변성、배사적정도、수량수감염시간연장이증가。HSV-2 LAT、당단백G(gG)기인PCR확증급전영결과증실병독재세포중적잠복급격활。LAT ORF-siRNA전염세포후，LAT ORF mRNA적표체수평재전염후24、36화48 h분별감소료39％、51％화60％。결론 억제LAT ORF후능구억제HSV-2 LAT기인적표체，위진일보연구LAT ORF재병독잠복감염복발중적작용제공료의거。
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.
