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单纯疱疹病毒2型潜伏相关转录体开放读码框3对Vero细胞凋亡的影响 被引量：4

Effects of herpes simplex virus 2 latency-associated transcript open reading frame 3 on the apoptosis in Vero cells
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摘要 目的研究单纯疱疹病毒2型（HSV-2）潜伏相关转录体（LAT）开放读码框3（ORF3）对顺铂诱导的非洲绿猴肾细胞（Vero细胞）凋亡的影响。方法构建重组质粒pEGPF—ORF3，并转染Vero细胞，逆转录聚合酶链反应（RT—PCR）验证目的基因的表达。顺铂诱导Vero细胞凋亡，通过荧光显微镜观察凋亡小体，Giemsa染色检测细胞核形态，噻唑蓝法（MTr法）检测细胞增殖，流式细胞仪分析细胞凋亡情况。实验结果采用SPSSl3．0进行分析，各组间两两比较采用单因素方差分析和t检验。结果成功克隆HSV-2333株ORF3基因，构建pEGFP—ORF3真核表达载体，RT—PCR验证该真核表达载体能在Vero细胞中高效表达。转染pEGFP—ORF3的Vero细胞经顺铂诱导凋亡后Giemsa染色，显示胞核蓝染，胞质淡浅，细胞形态正常。M．rr法分析显示，细胞增殖在转染pEGFP—ORF3组（2．56±0．21）与未经任何处理的正常对照组（2．66±0．13）比较，差异无统计学意义（P〉0．05），但高于顺铂诱导凋亡的正常对照组（1．65±0．11）和pEGFP—C2组（1．56±0．18），差异均有统计学意义（P〈0．05）；流式细胞仪分析细胞凋亡率，转染pEGFP—ORF3组（4．03％±1．04％）与正常对照组（2．13％±0．09％）比较，差异无统计学意义（P〉0．05），但低于空质粒pEGFP—C2组（19．45％±2．05％），且差异有统计学意义（P〈0．05）。结论HSV-2LATORF3在Vero细胞中具有抗顺铂诱导的凋亡作用。 Objective To explore the effects of herpes simplex virus 2 （HSV-2） latency-associated transcript open reading frame 3 （LAT ORF3） gene on Vero cells against cisplatin-induced apoptosis. Methods Recombinant plasmid enhanced green fluorescent protein-open reading frame 3 （named pEGFP-ORF3） was constructed and transfected into Vero cells; then, reverse transcription （RT）-PCR was performed to detect the expression of the target gene. Cisplatin of 3 mg/L was selected to induce the apoptosis in Vero cells. Cultured Vero cells were transfected with empty plasmid and induced by cisplatin （pEGFP-C2 group）, transfected with recombinant plasmid pEGFP-ORF3 and induced by cisplatin （pEGFP-ORF3 group）, only induced by cisplatin （cisplatin-induced control group）, or remained untreated （normal control group）. Subsequently, fluorescence microscopy was conducted to observe apoptotic bodies, Giemsa stain to observe the morphology of cell nuclei, methyl thiazolyl tetrazolium （MTY） assay to evaluate cell proliferation, and flow cytometry to assess cell apoptosis. Data were assessed by using SPSS 13.0 software, and statistical analysis was carried out by one-way ANOVA and t test. Results HSV-2 333 LAT ORF3 gene was successfully cloned. The eukaryotic expression plasmid for LAT ORF3 was constructed, and the expression of LAT ORF3 gene in Vero cells was confirmed by RT-PCR. Giemsa stain showed blue-staining nuclei and pale cytoplasm in recombinant plasmid-transfected and cisplatin-induced Vero cells with a normal shape. The value of cell proliferation （absorbance at 490 nm） by MTT assay was 2.56 ± 0.21 in pEGFP-ORF3 group, similar to that in the normal control group （2.66 ±0.13, P 〉 0.05）, but significantly higher than cisplatin-induced control group （1.65 ± 0.11, P 〈 0.05） and pEGFP-C2 group （1.56 ± 0.18, P 〈 0.05）. As far as the apoptosis rate was concerned, no significant difference was observed between pEGFP-ORF3 group and normal control group （4.03%± 1.04% vs. 2.13% ± 0.09%, P 〉 0.05）, but pEGFP-ORF3 group was statistically lower than pEGFP-C2 group （19.45%± 2.05%, P 〈 0.05）. Conclusion The transfected HSV-2 LAT ORF3 gene could protect Vero cells from cisplatin-induced apoptosis.
作者 杨慧兰 薛礼长 樊建勇 张发洲 龙朝钦 张玲
机构地区 广州军区广州总医院皮肤科
出处 《中华皮肤科杂志》 CAS CSCD 北大核心 2012年第3期186-190,共5页 Chinese Journal of Dermatology
基金 国家自然科学基金（30972666,81071401） 全军医学科学技术研究“十一五”计划（06J008）
关键词 疱疹病毒2型 人 开放读码框架 潜伏相关转录体 Vero细胞 顺铂 细胞凋亡 Herpesvirus 2, human Open reading frames Latency-associated transcripts Vero cells Cisplatin Apoptosis
分类号 R3 [医药卫生—基础医学]
  • 相关文献

参考文献12

  • 1Stanberry LR, Jorgensen DM, Nahmias AJ. Herpes simplex viruses 1 and 2. New York: Plenum Medical Book Company, 1997: 419-454.
  • 2Inman M, Perug GC, Henderson G, et al. Region of herpes simplex virus type 1 latency-associated transcript sufficient for wild-type spontaneous reactivation promotes cell survival in tissue culture. J Virol, 2001, 75(8): 3636-3646.
  • 3Li S, Carpenter D, Hsiang C, et al. Herpes simplex virus type 1 latency-associated transcript inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells following serum starvation by maintaining protein kinase B (AKT) levels. J Gen Virol, 2010, 91(Pt 4): 858-866.
  • 4Jin L, Carpenter D, Moerdyk-Schanwecker M, et al. Cellular FLIP can substitute for the herpes simplex virus type 1 latency- associated transcript gene to support a wild-type virus reactivation phenotype in mice. J Neurovirol, 2008, 14(5): 389-400.
  • 5Carpenter D, Henderson G, Hsiang C, et al. Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity. Microb Pathog, 2008, 44(2): 98-102.
  • 6Branco FJ, Fraser NW. Herpes simplex virus type 1 latency- associated transcript expression protects trigeminal ganglion neurons from apoptosis. J Virol, 2005, 79( 14): 9019-9025.
  • 7杨慧兰,白利利.单纯疱疹病毒2型潜伏相关转录体ORF2的表达及其抗凋亡作用[J].微生物学通报,2010,37(3):389-393. 被引量：5
  • 8Kranse PR, Ostrove JM, Straus SE. The nucleotide sequence, 5' end, promoter domain, and kinetics of expression of the gene encoding the herpes simplex virus type 2 latency-associated transcript. J ~irol, 1991, 65(10): 5619-5623.
  • 9Thomas SK, Lilley CE, Latchman DS, et al. A protein encoded by the herpes simplex virus (HSV) type 1 2-kilobase latency- associated transcript is phosphorylated, localized to the nucleus, and overcomes the repression of expression from exogenous promoters when inserted into the quiescent HSV genome. J Virol, 2002, 76(8 ): 4056-4067.
  • 10Thomas SK, Gough G, Latchman DS, et al. Herpes simplex virus latency-associated transcript encodes a protein which greatly enhances virus growth, can compensate for deficiencies in immediate-early gene expression, and is likely to function during reactivation from virus latency. J Virol, 1999, 73( 8 ): 6618-6625.

二级参考文献11

  • 1仕瑶慧,樊建勇,杨慧兰.潜伏相关转录体在单纯疱疹病毒中的作用[J].国际皮肤性病学杂志,2006,32(5):324-326. 被引量：5
  • 2Jennifer R Kent, Wen Kang, Cathie G Miller, et al. Herpes simplex virus latency-associated transcript gene function. Journal of NeuroVirology, 2003(9): 285-290.
  • 3Jin L, Carpenter D, Moerdyk Schauwecker M, et al. Cellular FLIP can substitute for the herpes simplex virus type 1 latency-associated transcript gene to support a wild-type virus reactivation phenotype in mice. J Neurovirol, 2008, 14(5): 389-400.
  • 4Carpenter D, Henderson G, Hsiang C, et al. Introducing point mutations into the ATGs of the putative open reading frames of the HSV-1 gene encoding the latency associated transcript (LAT) reduces its anti-apoptosis activity. Microb Pathog, 2008, 44(2): 98-102.
  • 5Francisco J Branco, Nigel W. Herpes simplex virus type 1 latency-associated transcript expression protects trigemihal ganglion neurons from apoptosis. J Virol, 2005, 79(14): 9019-9025.
  • 6Derfuss T, Arbusow V, Strupp M, et al. The presence of lytic HSV-1 transcripts and clonally expanded T cells with a memory effector phenotype in human sensory ganglia. Ann N YAcad Sci, 2009(1164): 300-304.
  • 7Shen W, Sa E Silva M, Jaber T, et al. Two small RNAs encoded within the first 1.5 kb of the herpes simplex virus typel (HSV-1) latency-associated transcript (LAT) can inhibit productive infection and cooperate to inhibit apoptosis. J Virol, 2009, 83(18): 9131-9139.
  • 8Everett H, McFadden. Poxviruses and apoptosis: a time to die. Curt Opin Microbiol, 2002(5): 395-402.
  • 9Daly CJ, Mcgrath JC. Fluorescent ligands and protein for the study of receptors. Phamacol Ther, 2003, 100(2): 101-118.
  • 10Mottk R, Osorio N, Jin L, et al. The bovine herpes virus-1 LR ORF2 is critical for this genes ability to restore the high wild-type reactivation phenotype to a herpes simplex virus-1 LAT null mutant. J Gen Virol, 2003(84): 2975-2985.

共引文献4

同被引文献33

  • 1Rajasagi NIC, Suryawanshi A, Sehrawat S, et al. Galectin-1 reduces theseverity of herpes simplex virus-induced ocular immunopathologicallesions[J]. Journal of immunology, 2012 ,188(9):4632-4643.
  • 2Boyd AS, Zwemer JP, Miller, JL,et al. Herpes simplex virus-inducedplasmacytic atypia [J]. Journal of Cutaneous Pathology, 2012,39(2):270-273.
  • 3Schouten JT,Schiffer JT. Equal HIV-1 Decay Kinetics in HSV-2-In-fected and HSV-2-Uninfected Clinical Trial Participants Treated WithAntiretroviral Therapy [J]. Journal of acquired immune deficiencysyndromes, 2012, 60(1):68-71.
  • 4Keating TM, Kurth AE, Wald A, et al. Clinical Burden of Herpes Sim-plex Virus Disease in People With Human Immunodeficiency Virus[J]. Sexually Transmitted Diseases, 2012, 39(5):372-376.
  • 5Barnabas RV, Celum C. Infectious Co-Factors in HIV-1 TransmissionHerpes Simplex Virus Type-2 and HIV-1: New Insights and Interven-tions[J]. Current HIV Research, 2012,10(3):228-237.
  • 6Szostek S, Zawilinska B, Kopec J, et al. Herpesviruses as possible co-factors in HPV-16-related oncogenesis [J]. Acta Biochimica Polonica,2009,56(2):337-342.
  • 7Jiang XZ, Chentoufi AA, Hsiang CH, et al. The Herpes Simplex VirusType 1 Latency-Associated Transcript Can Protect Neuron-DerivedCl300 and Neuro2A Cells from Granzyme B-Induced Apoptosis andCD8 T-Cell Killing[J]. J Virol, 2011,85(5):2325-2332.
  • 8Blather A, Raftery, MJ, Devi-Rao G, et al. Herpes Simplex Virus Type1 (HSV-l)-Induced Apoptosis in Human Dendritic Cells as a Resultof Downregulation of Cellular FLICE-Inhibitory Protein and ReducedExpression of HSV-1 Antiapoptotic Latency-Associated TranscriptSequences[J]. J Virol, 2010,84(2):1034-1046.
  • 9Du T, Zhou GY, Roizman B, et al. HSV-1 gene expression from reacti-vated ganglia is disordered and concurrent with suppression of latency-associated transcript and miRNAs [J]. PNAS, 2011, 108 (26):18820-18824.
  • 10Krause PR, Ostrove JM, Straus SE. The nucleotide sequence, 5, end,promoter domain, and kinetics of expression of the gene encoding theherpes simplex virus type 2 latency-associated transcript [J]. J Virol,1991,65(10):5619-5623.

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中华皮肤科杂志
2012年 第3期

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