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Aim:To construct a recombinant adenovirus that can simultaneously expressboth antisense ornithine decarboxylase(ODC)and adenosylmethionine decar-boxylase(AdoMetDC)and detect its inhibitory effect on the intracellular polyaminepool and colorectal cancer cell growth.Methods:A 205-bp cDNA of AdoMetDCwas reverse-inserted into recombinant pAdTrack-ODCas vectors and recombinedwith pAdEasy-1 vectors in AdEasy-1 cells.Positive clones were selected andtransfected into the packaging cell HEK293 after they were linearized by PacI.Green fluorescent protein expression was used to monitor the process of adenovi-rus packaging.The ODC and AdoMetDC protein levels were identified by west-ern blotting,and intracellular polyamine content was detected by reverse-phasehigh performance liquid chromatography.A viable cell count was used to deter-mine the growth of HT-29 cells with or without exogenous polyamine.Results:Sequencing confirmed that AdoMetDC cDNA was successfully ligated into thepAdTrack-ODCas vector.GFP expression in 293 cells during virus packing andamplification was observed by fluorescence microscopy.Western blotting dem-onstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas,and consequently 3 kinds of polyamine(putrescine,spermidineand spermine)were reduced to very low levels.HT-29 cell growth was signifi-cantly inhibited as compared with control conditions,and growth arrest was notreversed by exogenous putrescine.Conclusion:The successfully constructedrecombinant adenovirus,Ad-ODC-AdoMetDCas,blocked polyamine synthesisand has therapeutic potential for treating colorectal cancer in vitro.