Abstract:

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 （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.

Key words: Anti-apoptotic function