Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide

ObjectiveTo construct the recombinant adenovirus expressing small RNA of rats caspase-3 and observe the down-regulation effect of caspase-3 in neurons induced by lipopolysaccharide(LPS) in vitro.MethodspShuttleH1-siCas3 containing Oligo DNA of the targeting sequences and pEGFPC1-Cas3 containing caspase-3 and EGFP sequences were constructed respectively. pShuttleH1-siCas3 and pEGFPC1-Cas3 were co-transfected to the 293 cells by liposomes to determine interfering efficacy by flow cytometry. pShuttleH1-siCas3 was linearized and transformed into E. coli BJ5183 cells containing backbone plasmid pAdEasy-1. The recombinant plasmid was transfected into 293 cells to package the adenovirus Ad-siCas3. The titers of adenovirus were determined by the specific 50% tissue culture infection dosage method. After virus infected the cultured hippocampus neurons, LPS-induced apoptosis and caspase-3 mRNA expression were observed.ResultsIt was identified that the sequence of target gene was correctly inserted into the genome of virus. The expression of green fluorescence protein was reduced by pShuttleH1-siCas3 in 293 cells. The titer of recombinant adenovirus was 1.06×1010 pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed.ConclusionThe recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.