PTCA後再狭窄に対する遺伝子治療の試み -アンチセンス技術の応用と遺伝子導入-

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  • Prevention of Restenosis After Angioplasty : Efficacy of Antisense Strategy Against Cell Cycle Regulatory Genes

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To develop an effective strategy to prevent neointimal formation after angioplasty injury, we have identified cell cycle regulatory proteins as targets for inhibition using antisense oligonucleotides (ODN). In addition, we have developed a novel intraluminal molecular delivery method that employs the protein coat of the Hemagglutinating virus of Japan (HVJ) complexed with liposomes, to enhance the efficiency of cellular uptake and the stability of antisense oligonucleotides while minimizing non-specific toxicity. Phosphorothioate ODN are complexed with liposomes and the protein coat of the inactivated HVJ. This method results in a more rapid cellular uptake and a ten-fold higher transfection efficiency of plasmids or ODN than lipofection or passive uptake methods. HVJ-liposome complex containing ODN was incubated within the rat carotid artery for 10 minutes immediately after balloon injury. First, we examined the effect of antisense cdc (cell division cycle) 2 kinase, proliferating cell nuclear antigen (PCNA), cdk (cyclin dependent kinase) 2 kinase and cyclin B1 ODN on neointimal formation after balloon injury in the rat carotid artery model. Reverse transcription PCR in antisense ODN (PCNA and cdc 2 kinase) transfected vessels showed a marked decrease in cdc 2 and PCNA gene expression as compared to that in sense control ODN transfected vessels. The measurement of DNA synthesis as assessed by bromodeoxyuridine index and DNA content revealed a decrease in antisense PCNA and cdc 2 kinase ODN treated vessels on day 4 after injury as compared to sense ODN treated vessels. Transfection with either antisense cdc 2, cdk 2 kinase and cyclin B1 ODN alone resulted in a partial inhibition of neointimal formation. We then evaluated the strategy of combined antisense ODN to achieve complete inhibition. The combination of antisense ODN directed against both cdc 2 kinase and PCNA inhibited completely neointimal formation at 2 weeks after angioplasty. Moreover, the inhibitory effect of this antisense ODN combination on neointimal formation persisted up to 8 weeks after a single transfection. The combination of antisense cdc 2 kinase and cyclin B1 ODN, or antisense cdc 2 and cdk 2 kinase ODN, also resulted in near complete inhibition of neointimal formation. The presistent inhibition of neointimal formation after balloon injury was also observed by the combination of antisense cdc 2 kinase and cyclin B1 ODN. The present study documents that a single intraluminal molecular in vivo delivery of antisense cell cycle regulatory genes ODN results in a inhibition of neointimal formation in the rat carotid balloon injury model as a model of gene therapy. Interestingly, our results reveled that multiple blockade of cell cycle regulatory genes by antisense ODN are necessary to achieve the complete inhibition of neointimal formation after angioplasty.

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