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.
ABSTRACT
Objective To investigate the effect of photodynamic therapy with chlorin e6 (Ce6) and 5-aminolevulinic acid (5-ALA) on mouse model of malignant melanoma. Methods The mouse model of malignant melanoma was established by injecting 0.1 ml A-375 cells (about 2?10 6 cells) under the right hind leg of BALB/c-6 mice,and that the yellowish white node appears at injection site proves the successful model. Twenty-four of 27 successful mouse models were irradiated at the tumor site with semiconductor laser (wavelength 652 nm) with a total dose of 100 J/cm 2 . Before laser exposure,the mice were treated with 10% 5-ALA by topical compress for 2 h or 7.5 mg/kg Ce6 by intraperitoneal injection for 1 hour or 5-ALA topical application combined with intraperitoneal injection of Ce6 (n=6 in each group). Another six mice as control only underwent PDT. One week after PDT,the mice were killed,the tumor mass was peeled off and weighed,whether the metastasis occurred or not was detected,and the tumor,liver,spleen,lung,kidney were sent to histopathological examination. Results The tumor weight in 5-ALA group,Ce6 group,and the combined group had significant difference as compared with control group (P0.05). The dehydration and scab formation and necrosis could be seen in tumor sites at 1 week after PDT. The cell collapse and necrosis,subdermal thrombosis and cell outline clouding could be observed by histopathological examination. Metastasis of melanoma were found in 5-ALA group,Ce6 group,and the combined group. Conclusion PDT with Ce6 and 5-ALA could kill the malignant melanoma effectively in animal experiment but could not affect the metastasis of melanoma.