Effection On Early Cardiac Allograft By Adenoviral Vector-mediated Human Hepatocyte Growth Factor Gene Transfection In Rats

Objective To investigate the effect on early cardiac allograft by adenoviral vector-mediated human hepatocyte growth factor(HGF) gene transfection in rats. Methods Construction of the replication deficient recombinant adenoviruses (Ad-HGF) of human HGF; Simplified Cervical cardiac allograft model of rat Were Suceessfully established by modified cuff vessel anastomosis. Rats donor Hearts coronary arteries were perfused ex vivo with Stanford University solution containing 2.5×1010plaque-forming units/ml of Ad-HGF, then implanted in the necks of rat recipients. Then the allografts were collected ,gene product expression in tissue was quantified by immunoassay,reverse transcription-Polymerase chain reations(RT-PCR), enzyme-linked immunosorbent assay (ELISA) analysis and immunohistochemical staining. The number of inflammatory cell infiltration and myocardial cell apoptosis in cardiac allografts was detected.Results Ad-HGF and cervical cardiac allograft model of rat were suceessfully established. Ad-HGF transfer into cardiac allograft and express efficiently and stably in vivo. HGF can decrease inflammatory reaction of cardiac allograft, improve myocardial protection from ischemia/reperfusion injury and antiapoptotic action after transplantation.Conclusion It can significantly effected on early cardiac allograft by adenoviral vector-mediated human HGF gene transfection in rats.PART 1Model of cervical heterotopic heart tansplantation in rats Objective To establish the authetic model of cervical heterotopic heart transplantation in modified Heron,s technique in rats. Methods The Wistar rat donor,s ascending aorta and Pulmonary artery were anastomosed to SD rat recipient right common carotid artery and external jugular vein respectively with a self-made"sleeve"anastomosis.Result 85 of 86 successful transplantations have been performed by single surgeon ,were done with negligible operative risk by cuff vessel anastomosis ,except of 1 donor's heart bleeding. Conclusion This modified technique was a simple,economical,practicable,reliable and high reproducible model,can be operated by surgeons with minimal training in microvascular surgery,and can be applied to various transplantation immunological studies.PART 2Construction of a Recombinant Adenovirus Carrying Human Hepatocyte Growth Factor GenObjective To investigate construction of a recombinant adenovirus carrying human hepatocyte growth factor gene.Methods To identify HGF gene,the recombinant Aadenovirus Ad-HGF was construted by us in Ad5Easy system based on the homologous recombination in bacteria.Rasults The adenovirus Ad-HGF was construted by homologous recombination bacteria using Ad5Easy system.Conclusion Ad-HGF was successfully construted by homologous recombination in bacteria,it is a base to research the gene transfer of HGF.PART 3Effection of Adenoviral Vector-mediated Human Hepatocyte Growth Factor Gene on the acute rejection reactions after allogenic cardial transplantation of rats Objective Intracoronary infusion of adenoviral vector-mediated human HGF gene transfer effects on the acute rejection reactions after allogenic cardial transplantation of rats. Methods In a cervieal heterotopic cardiac transplantation model by modified technique,after harvest,the donor hearts'coronary arteries were perfused ex vivo with Stanford University solution containing 2.5×1010pfu/ml Ad-HGF at 4°C for 30 minute(group A),then implanted in the necks of rat recipients. As controls,other hearts were perfused with Stanford University Solution containing 2.5×1010pfu/ml adenoviral vector(Ad) alone did not delivered that gene(grou B) or with nothing but Stanford University solution(grou C) for the same period. The allografts were collected after 1,3,7,14 days.Gene product expression in tissue was quantified by immunoassay,reverse transcription-Polymerase chain reations(RT-PCR) and visualized and localized by immunohistochemical staining. the heart rate were recorded everyday to find which group heart have the longest survival time. The allograft myocardium were stained with HE to observe the strength of immunologic rejection.The expression of IL-2,IL-10 and IFN-γwere detected. The ultrastructure and myocardial apoptosis of the graft tissues were observed . Results 1 .The survival durations of the grafts in group A were obviously longer than those of the other two groups (P<0.01). 2. After transplantation transgene expressions of HGF in group A were detected by means of RT-PCR.Immunohistochemical examination of Ad-HGF infected grafts (group A) confirmed successful gene transfer expression in myocardial cells.3. The expression of IL-2, IFN-γin group A were lower than in the other two groups but the expressions of IL-10 in group A were the opposite. 4. The apoptotic index of myocardial cells in group A were lower than those in group B and C (P<0.05). Ultrastructural alterations indicated that injury of grafts in group A were less severe than in group B and C. Conclusion 1.These data demonstrate that intracoronary gene transfer of adenoviral vector-mediated human HGF gene to cardiac allografts is efficient and effectively in rats.2. HGF can decrease inflammatory reaction of cardiac allograft, improve myocardial protection from ischemia/reperfusion injury and antiapoptotic action after transplantation.3. Intracoronary infusion of adenoviral vector-mediated human HGF gene transfer significantly effects on the acute rejection reactions after allogenic cardial transplantation of rats.