The growth-inhibitory effect of recombinant ING4-adenovirus on MG-63 osteosarcoma cells.

Objective:To construct recombinant adenovirus harboring humanarized mouse ING4 gene and to study its growth-inhibitory effects on osteosarcoma cells.Methods:In order to obtain the recombinant ING4 Adenovirus vector,site-specific mutagenesis technique was used,and pcDNA3.0-mING-4 was used as the template.Then the recombinant Ad-ING4 was obtained by gene recombination and in vitro packaging technique.Osteosarcoma cells were infected with Ad-ING4 and RT-PCR was used to detected the transcription of ING4 in the cells.Cell growth inhibition was detected by MTT assay.Morphologic changes were observed by light microscope and fluorescence microscope.The cell cycle and apoptosis were detected by flow cytometry,and morphologic changes of the infected cells were observed under laser scanning confocal microscopy(LSCM).In addition,RT-PCR was used to detected the transcription of Bax and Bcl-2.Results:Ad-hING4 were proved in correct construction by gene sequence and PCR results.Expression of hING4 was identified by RT-PCR.By microscope and flow cytometry displayed the apoptosis of the osteosarcoma cells positively expressing ING4.The apoptotic body in the cells was shown by laser scanning confocal microscopy(LSCM).Additionally,the mRNA of Bax was up-regulated and mRNA of Bcl-2 was down-regulated in the cells by RT-PCR.Conclusion:Ad-ING4 can inhibit the growth of MG-63 cells and induce cell apoptosis,which may result in changing Bax and Bcl-2 expression.