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(MTT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SPSS13.0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-2 333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.MTT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-2 LAT ORF3在Vero细胞中具有抗顺铂诱导的凋亡作用.

abstractsObjective 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 (MTT) 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.