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

Abstract

Objective To 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. Methods pShuttleH1-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. Results It 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 1.06×10 10 pfu/ml. After virus infection, caspase-3 mRNA was greatly reduced and neurons apoptosis was suppressed. Conclusion The recombinant adenovirus expressing rats caspase-3 siRNA were successfully constructed, which may probably be further used in pain therapy by its anti-apoptosis effect.