Anti-Tumor Effects Of Recombinant Adenovirus With HN Genes Co-Regulated By MTERT And MTyr Promoters On B16 Nude Mice Model

Melanoma is a malignant tumor with a very high mortality rate.It is common in veterinary clinics in elderly animals.The incidence rate of it gets higher year by year within the scope of world.It is characterized by high degree of malignancy,rapid progress,and easy distant metastasis.It is a malignant tumor that has strong resistance to conventional treatment drugs and is rather troublesome in clinical treatment.Gene therapy has become a research hotspot.As a superficial tumor,melanoma is very suitable for the observation and research of gene therapy.In the previous study,the group selected the hemagglutinin-neuraminidase(HN)gene of the Newcastle Disease Virus(NDV)as the treatment gene for melanoma.Homologous recombination of mouse telomerase(m TERT)promoter,mouse melanoma tyrosinase(Tyr)specific promoter core fragment and HN gene with adenovirus skeleton was performed.Tumorspecific and tissue-specific recombinant adenovirus Ad-m TERTp-m Tyrp-HN was successfully constructed.Recombinant adenovirus Ad-m TERTp-Tyrp-HN,which was constructed in the early stage was used in this study.The titers of Ad-HN,Ad-m TERTp-HN,Ad-m Tyrp-HN,Ad-m TERTpTyrp-HN and Ad-GFP were 1012.5TCID50/m L,1011.75TCID50/m L,1012.375 TCID50/m L,1012.875TCID50/m L and 1011.375TCID50/m L,respectively.Recombinant adenovirus Adm TERTp-Tyrp-HN was transfected into melanoma cell,cck-8 method was used to monitor cell survival rate by observing cell growth status and expression of green fluorescent protein.The results showed that Ad-m TERTp-Tyrp-HN had dose-dependent and time-dependent effects on tumor inhibition of B16 cells cultured in vitro.Through AO/EB staining,DAPI staining,and crystal violet,the morphological method of recombinant adenovirus inhibiting tumor was clearly determined.The results showed that recombinant adenovirus could induce apoptosis to varying degrees.The AO/EB staining results show that most of the control cells of the uninfected virus showed uniform green(ie,most of them were normal cells),and only a small amount of cells with early apoptosis were orangered;CPE cells transfected with Ad-HN,Ad-m TERTp-HN,Ad-m Tyrp-HN,Ad-m TERTp-TyrpHN and Ad-GFP were transfected.After DAPI staining,CPE was clearly seen in the cells of the four groups infected with adenovirus,while the cells of the control group were uniformly bright blue and no nuclear fragmentation was observed.The migration and invasion ability of recombinant adenovirus to B16 cells were detected by cell scratch test and cell invasion test.The cell apoptosis was detected by scanning electron microscopy,the cell membrane was shriveled,the cell membrane was foamed or “budded”,and many bulbous vesicles were formed,which were in the form of bubble,multi-process or cloud,and the morphology was highly irregular,and smooth pits on the cell surface were observed,and the cell lesions were obvious.In this study,BALB/c mouse melanoma tumor model was constructed,and the inhibition of Ad-m TERTp-Tyrp-HN on B16 cell was investigated by intravenous injection and intratumor injection.Although the tail intravenous injection group could not eliminate the tumor,it made the tumor grow slowly.After 48 h injection of the tail vein,frozen sections could be observed green fluorescence at the tumor tissue;Real time fluorescence quantitative PCR results showed that Ad-m TERTp-Tyrp-HN can multiply the expression of m RNA in caspase-3,p21,and p53.The intra-tumor injection group showed that Ad-m TERTp-Tyrp-HN could prolong the median survival period of the mouse melanin model,which was longer than that of the cisplatin treatment group,without causing obvious damage to liver,kidney,spleen and other tissues,and without weight loss.In vivo imaging results showed that the recombinant adenovirus could still be expressed in the tumor tissue and was obviously distributed after 96 h intratumor-injection.The results of frozen section showed that the fluorescence intensity of Ad-m TERTp-Tyrp-HN was higher than that of the control group.The expression levels of caspase-3,p21 and p53 proteins were up-regulated by recombinant adenovirus by Western blotting,which was consistent with the fluorescence quantitative results.TUNEL assay showed that Ad-m TERTpTyrp-HN brown area accounted for 91%;Immunohistochemical results showed that the positive rates of caspase-3,p21 and p53 protein in the experimental group were higher than those in the control group(p<0.05).The experimental results of intravenous and intratumor injection showed that although both different treatments had the effect of inhibiting the growth of solid tumors,the treatment effect of Ad-m TERTp-Tyrp-HN injection was more prominent.In summary,Ad-m TERTp-Tyrp-HN had tumor suppressor effect in vitro,and had a targeted nature.It showed a clear anti-tumor treatment potential and was expected to provide effective treatment strategies for the clinical treatment of melanoma.It will lay the foundation for the treatment of diseases with recombinant adenovirus.