| At the present time, the most crucial obstacle in transplantation is rejective reaction and ischemia-reperfusion injury. The introduction of functional genes into organ grafts before transplantation has been an attractive approach to modulate deleterious posttransplant events. The range of potential therapeutic applicants includes amelioration or prevention of host versus graft immune reactions and reduction of graft ischemic and preservation injury. Effective gene therapy requires a reliable method of gene transfer that efficiently inserts target genes to the parenchyma! and/or nonparenchymal cells in grafts to produce required levels of proteins. Viral vectors, especially replication-defective adenoviruses, have been favored because of their ability to readily infect nonproliferating cells. Although adenoviral-mediated recombinant gene expression has been limited to a short period after gene transfer, this approach may provide advantages in modifying the critical early posttransplant period when intensive ischemic graft injury occur.PART I:Objective: To introduce an animal model of modified cuff technique orthotopic of liver transplantation in rat.Methods: 150 cases of rat orthotopic liver transplantation were performed by using the two-cuffed technique with some modification in graft harvesting, recipients anesthesia, the anastomoses of suprahepatic vena cava and the perioperative treatment.Results: Of the last 30 cases , the mean time of anhepatic period was 13min, and the mean operation time was 45min. The 24 hours and 2 week survival rates were 97% and 90% respectively.Conclusions: The modified two-cuffed methods is convenient and easy to duplicated. It can enhance the stability and survival rate of rat orthotopic liver transplantation models.PART II:Objective: To estimated the effect of heat preconditioning of liver grafts on the transplant survival rate and on apoptosis of hepatocytes as well as sinusoidal endothelial cells (SEC) in a rat model of liver transplantation.Methods: Donor rats of the heat shock (HS) group were subjected to heat preconditioning 48 hrbefore graft harvest. The liver grafts from the HS group and control (C) group were preserved in Ringer's lactate solution for 4 hour and transplanted orthotopically. Three hours after reperfusion , the recipients were sacrificed , serum ALT> AST. LDH were measured and HSP7(h TNF- a , bcl-2 levels were estimated by immunohistochemistry, apoptosis of the hepatocytes and SEC was analyzed by TLJNEL.Results: HSP70 expression was detected not only in hepatocytes but also in SEC. In the 4 hour preservation model, the 1-week survival rate was 62.5% in the HS group and 25% in the C group. Serum ALT. AST. LDH levels in the HS group were significantly lower than those in the C group at 4 hr after reperfusion. Immunohistochemistry study showed the TNF- a level in C group was higher than in HS group , whereas the difference of bcl-2 level between the two group was not distinct. In TUNEL analysis, the number of positive SEC in the C group was markedly increased compared with the HS group.Conclusions: Heat preconditioning of the graft improved the survival rate of the liver transplants. Induction of HSP70 in hepatocytes as well as in SEC might attenuate preservation-reperfusion injury from TNF- a induced apoptosis .PART III:Objective: To construct a recombinant adenovirus vectors (Ad.CMV-HSP70), in which the HSP70 gene are under the control of the CMV promoter.Methods .- Human heat shock protein 70(hHSP70) cDNA were cloned into adenovirus shuttle vector PDC312-CMV by standard procedure. The recombinant adenoviral plasmid PDC312-CMV-HSP70 was identified and the correct clones containing target gene in right direction were selected. PDC312-CMV-HSP70 and pBGHAE3, each bearing a loxP site and the latter containing a ere site , were cotransferred into the adenoviral packaging 293 cell by lipofectamine mediated gene transfer method to recombinant and pack the virus. After identified, the desired Ad vectors were purified by density gradient u... |
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