Antitumor Effects Of The Tumor Specific Adenovirus

Tumor is a kind of disease which can threaten human health seriously.Because of its specific biological behaviour,operation,radiotherapy and chemotherapy can not increase survival quality of patients and prolong their life.Yet,many of these deaths can be avoided. Over 40%of all malignant tumours can be prevented.Others can be detected early,treated and cured.Even with late stage cancer,the suffering of patients can be relieved with good palliative care.With the development of molecular biology and hnmunology,we realized that tumor ia a kind of disease related gene,abnormality of normocellular growth control and mutation of apoptosis regulatory gene can result in alteration of function,then tumor cell get the ability of metastasis and infiltration,proliferate minute primary lesion gradually.In this study,Apoptin and HN gene were placed downstream of CMV promoter of shuttle plasmid of RAPAd.I system.The recombined recombinant adenovirus named Ad-HN and Ad-Apoptin.The above shuttle plasmids of dual specific recombinant adenovirus and genome skeleton of RAPAd.I system were co-transfected into AAV-293 cells.The recombinant adenoviruses Ad-HN,Ad-Apoptin and Ad-EGFP were obtained by homologous recombination.The expression of Apoptin,HN of the recombinant adenovirus was identified by RT-PCR,western blot and indirect immunofluorescence.The results showed that the foreign genes expressed effectively in HepG-2 cells.HN gene has activity of neuraminidase, hydrolyze sialic acid in the surface of tumor cell,expose tumour-cell surface antigen, strengthen identification and killing of tumor cell through immune system.HN can induce IFN,increase recognization of tuor cell by CTL,correct functional defect of immune system partly.Apoptin can induce apoptosis of tumor cell,its function don not depend on p53 and bcl-2,Apoptin can not induce normal cell.The anti-tumor effects of Ad-Apoptin,Ad-HN and Ad-EGFP on HepG-2 cells were detected by MTT.The results showed that,except for Ad-EGFP,Ad-Apoptin and Ad-HN killed HepG-2 cells in varying degrees.In the certain time frame,the suppression rates of Ad-HN and Ad-Apoptin on HepG-2 cells were heightened with the stepping up of infective dose.In the definition of infection doses,the suppression rates of Ad-HN and Ad-Apoptin on HepG-2 cells were improved with the extending of infection time.By using observation of light microscope,AO/EB staining,DAPI staining,AnnexinⅤstaining and caspase activity detecting,we approached the decease pathway of HepG-2 cells reduced by the dual specific recombinant adenoviruses.The results showed that the recombinant adenovirus induced apoptosis of HepG-2 cells.C57BL/6 mice model bearing H22 hepatoma was constructed by transplanting H22 hepatoma cell into the right hind limb of the mice and the anti-tumor effects of the recombinant adenoviruses described above were observed through this model.The results showed that,compared with saline and control groups,experiment groups displayed varying degree anti-tumor effects.The suppression rates of Ad-HN(44.65%) and Ad-Apoptin (33.55%),it is higher than that of other groups.The survival rates of Ad-HN,five of six mice survived(survival rate was 83.33%),was significant higher than saline-,Ad-EGFP-,Ad-Apoptin-treated groups.The survival rates of Ad-Apoptin-treated groups were 50%,and the survival rates of Ad-EGFP- treated groups was 33.33%.The electron microscope observation of tumor cell showed nucleic condensation and localization,chromatin margination,transfected tumor cells showed typical apoptosis.The results of the.detection of cytokine of Th1/Th2 showed that the recombinant adenovirus up-regulated the level of some cytokines.The level of IL-2 and IFN-γof mice of Ad-HN-treated groups was much higher than other groups.This result illustrated that Ad-HN lead the immune response to Th1 dominant.On the other hand,perhaps,because of the immunostimulation of adenovirus itself,the levels of some cell factors of the mice of Ad-Apoptin-treated groups were up-regulated.In summary,the dual specific anti-tumor recombinant adenovirus constructed by using RAPAd.I system basing on specific anti-tumor gene Apoptin/HN in the study,which has tumor specific restrain abilities,can suppress HepG-2 cells and prolong life span of animal model effectively.The researches can provide a useful platform for the exploration of more safer,specific,effective anti-tumor medicine.