Effect Of Transforming Heat Shock Protein70 Gene Transfer On Angionosis Of Cardiac Allogeneil Graft

PART 1Construction of Recombinant Adenovirus Mediated Heat Shock Protein70Objective To investigate construction of recombinant adenovirus containing heat shock protein 70. Methods to identify HSP70,the recombinant adenovirus AD5–HSP70 was constructed by us in Ad5 Easy system based on homologous recombination in bacteria.Results the adenovirus Ad5-HSP70 was quickly constructed by homologous recombination bacteria using AdEasy system. Conclusion Ad5-HSP70 can be successfully constructed by homologous recombination in bacteria ,which lay the foundation for further research for gene transfer of HSP70PART 2Modification of Heron's technique for cervical heterotopic cardiac transplantationObjective This study modifed Heron's technique for cardiac transplantation in rats by cuff vessel anastomosis in some aspects and successfully established the simplified model of cervical cardiac transplantation from wistar donor to SD rat recipients. Methods The wistar donor 's ascending aorta and pulmonary artery were anastomosed to SD rat recipient's right common carotid artery and external jugular vein respectively with a self-made "sleeve" anastomosis. The modified cuff technique of heterotopic grafting is described in detail. Results 95 consecutive successful transplantations have been performed by single surgeon were done with negligible operative risk. There was neither anastomosis leakage nor vessel obstruction. The total time of surgical procedure were 45 to 60 minutes. The new technique allowed vascular anastomoses to be completed in 2 to 5 minutes, the total cold ischemia time for donor heart was 15 minutes in average. Conclusion This modified heron's 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 3Effect of Transforming heat shock protein70 Gene Transfer on angionosis of Cardiac allogeneil graftObjective The aim of the current work was to study the effect of recombinant adenovirus vector-mediated HSP70 (Ad.HSP70) gene transfer on cardiac allogeneil graft. Methods In a cervical heterotopic cardiac transplantation model by cuff technique, after harvest, wistar rat donor hearts' coronary arteries were perfused ex vivo with HTK solution containing 3.8×1010 VP/mL of donor heart of Ad.HSP70 at 4℃for 45 minute (group 3), then implanted in the necks of immunosuppressed rat recipients. As controls, other hearts were perfused with HTK Solution containing 3.8×1010 VP/Ml of donor heart adenoviral blank-vector (Ad) not delivered that gene (group 2) or with virus-free HTK solution (group 1) for the same period by the same method. The allografts were collected after one month. Gene product expression in tissue was quantified by immunoassay, reverse transcription-polymerase chain reations (RT-PCR) and visualized and localized by immunohistochemical staining. Results RT-PCR demonstrated the transcription of exogenous HSP70 gene in the allografts. Immunohistochemical examination determination of Ad.HSP70-infected grafts (group 3) confirmed successful HSP70 gene transfer expression in perivascular myocardial cell and coronary capillaries. Expression Level of foreign gene significantly higher in Ad.HSP70-infected grafts compared with other two groups (P<0.05). The number of inflammatory cell infiltration in cardiac allografts of Ad.HSP70-infected group was less than that of other groups (P<0.01). Over expression of exogenous HSP70 gene increase the expression of Bcl-2 and decrease the expression of VCAM-1 and NF-KB attenuated macrophage and nature killer cell infiltration in the allografts and protect allograft endothelial cell.Conclusions These data demonstrate that intracoronary gene transfer of recombinant adenovirus vector encoding HSP70 to cardiac allogeneil graft is efficient and effectively and protect allograft endothelial cell. Recombinant adenovirus vectors can induce localized expression of such potential therapeutic agent within donor allografts that would make this approach widely clinically applicable.