Adenoviral vector mediated-expression of caspase-3 siRNA on apoptosis induced by lipopolysaccharide |
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Authors: | Wu Feixiang Yu Weifeng Yuan Yang Miao Xuerong Xu Xuewu Huang Shengdong Sun Yuming |
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Institution: | [1]Department of Anesthesiology, Eastern Hepatobiliary Hospital, Second Medical Military University, Shanghai 200438, China [2]Institute of Thoracic Cardic Surgery, Changhai Hospital, PLA, Shanghai 200433, China |
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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 eytometry, 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 1.06×1010 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. |
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Keywords: | Caspase-3 RNA interference Adenovirus Apoptosis Neuron |
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