Basic study for gene therapy by

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[Purpose]As a factor of anastomotic stricture after bypass surgery using an artificial graft, involvement of platelet-derived growth factor beta : PDGF beta chain, has been reported. PDGF beta chain is a powerful cell growth factor for smooth muscle cells, and such cells at the anastomotic site have been reported to show high-level expression of PDGF beta chain receptor. The purpose of this study was to clarify the effect to avoid stricture, through gene introduction of the extracellular region of receptor EX, which inhibits intimal proliferation, into the anastomotic site.[Subjects and method]We grafted expanded polytetrafluoroethylene artificial blood vessels, three millimeters in diameter, to rat aorta.Experiment 1.We administered a replication-deficient recombinant adenovirus containing the E.coli LacZ gene(AxLacZ), at levels of 1x10^8, 1x10^9, 1x10^<10>, and 1x10^<11>pfu/ml, into the anastomotic blood vessels, (1)from the adventitial side, and (2)from the lumen. Two weeks after th … More e surgery, the introduction rates of E.coli LacZ gene were measured by X-Gal staining.Experiment 2.Using the method that showed the best introduction rate in Experiment 1, we administered a replication-deficient recombinant adenovirus containing the gene encoding EX(AxPDGFXR) into the anastomotic sites of the artificial vessels. Two weeks after the surgery, we determined the protein concentration of AxPDGFXR and mRNA expression. One month after the surgery, we conducted HE staining, and immunohistological staining for von Winebrand factor and Ki-67 antigen, to assess arterialization and the stricture rates of the anastomotic sites. We performed DNA fragmentation using the TUNEL method, and sought the rate of apoptosis.[Result]Experiment 1.Administration from the adventitial side of the anastomotic vessel at 1x10^<11> pfu/ml showed the best introduction efficiency.Experiment 2.EX protein expression and mRNA were identified at anastomotic artery. In the AxPDGFXR group, arterialization and cell growth of vessels immunohistologically stained for von Winebrand factor and Ki-67 antigen, were restricted, and the stricture rate was also significantly lower, compared with the control group. No apoptosis was found.[Conclusion]An experiment revealed that local expression of the extracellular region of platelet-derived growth factor receptor restricts intimal thickening of the artificial vessel anastomotic site. If effectiveness and safety of this method are established, clinical application to micro blood vessel grafting will become possible, and remarkable improvement of the patency rate can be expected. Less