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目的:研究单纯疱疹病毒2型潜伏相关转录体(LAT)开放读码框(ORF)对凋亡诱导剂诱导Vero细胞凋亡的影响。方法:PCR扩增LAT ORF1、ORF2、ORF3片段,构建重组质粒pEGFPORF1、pEGFP-ORF2、pEGFP-ORF3,重组质粒pEGFP-ORF1、pEGFP-ORF2、pEGFP-ORF3转染Vero细胞;RT-PCR鉴定ORF1、ORF2、ORF3的表达。用凋亡诱导剂放线菌素D、顺铂、5-氟尿嘧啶(5-FU)诱导Vero细胞凋亡,通过荧光显微镜观察细胞形态学的改变,MTT检测细胞活性,流式细胞术检测细胞凋亡率。结果:双酶切和测序确认pEGFP-ORF1、pEGFP-ORF2、pEGFP-ORF3构建成功,RT-PCR表明这3个真核表达载体能在Vero细胞中高效表达。转染各种pEGFP-ORF的Vero细胞经凋亡诱导剂诱导后,细胞形态正常。MTT结果表明转染了各种重组质粒pEGFP-ORF的Vero细胞经凋亡诱导剂诱导后Vero细胞活性与未经任何处理的正常对照组相比,差异无统计学意义(P>0.05),但高于凋亡诱导剂诱导凋亡的Vero细胞组及与转染空质粒pEGFP-C2,且经凋亡诱导剂诱导的Vero细胞组,差异具有统计学意义(P<0.05)。流式细胞术结果表明,转染各种重组质粒pEGFP-ORF且经凋亡诱导剂诱导凋亡组与正常对照组凋亡率差异无统计学意义(P>0.05),而显著低于转染空质粒pEGFP-C2且经凋亡诱导剂诱导凋亡组,差异有统计学意义(P<0.05)。结论 HSV-2 LAT ORF对凋亡诱导剂诱导的Vero细胞的凋亡具有抵抗作用。
Objective: To investigate the effect of herpes simplex virus 2 (LAT) open reading frame (ORF) on the apoptosis of Vero cells induced by apoptosis inducer. METHODS: The recombinant plasmid pEGFPORF1, pEGFP-ORF2 and pEGFP-ORF3 were amplified by PCR. The recombinant plasmid pEGFP-ORF1, pEGFP-ORF2 and pEGFP-ORF3 were transfected into Vero cells. The expression of ORF1, ORF2, ORF3 expression. Apoptosis of Vero cells was induced by actinomycin D, cisplatin and 5-fluorouracil (5-FU). Cell morphology was observed by fluorescence microscope. Cell viability was detected by MTT assay. Flow cytometry Death rate. Results: Double enzyme digestion and sequencing confirmed that pEGFP-ORF1, pEGFP-ORF2 and pEGFP-ORF3 were successfully constructed. RT-PCR indicated that these three eukaryotic expression vectors could express efficiently in Vero cells. The Vero cells transfected with various pEGFP-ORFs were induced by apoptosis inducer and the cell morphology was normal. The results of MTT assay showed that there was no significant difference in the Vero cell viability between Vero cells transfected with various recombinant plasmids pEGFP-ORF and normal control without any treatment (P> 0.05) Which was higher than Vero cells induced by apoptosis inducers and Vero cells transfected with empty plasmid pEGFP-C2 and induced by apoptosis inducers. The difference was statistically significant (P <0.05). The results of flow cytometry showed that there was no significant difference in apoptotic rate between the recombinant plasmid pEGFP-ORF transfected with apoptosis-inducing agent and normal control group (P> 0.05), but significantly lower than that of transfection The plasmid pEGFP-C2 was induced by apoptosis inducer, the difference was statistically significant (P <0.05). Conclusion The HSV-2 LAT ORF is resistant to apoptosis induced by apoptosis inducer in Vero cells.