Immunological Research On The Transplantation Of Cryopreserved Allogeneic Osteoarticular With Vessels

Objective: This experiment aims to observe the status of rejection reactions after transplantation through examination on the total number of T cells in the plasma, the CD4+/CD8+ proportion, and the changes of CD25+ cells before and after the operations with short-term application of Cy A like the transplantation of the allogeneic knee joints of the SD rat choke vessels that had been frozen and stored, the transplantation of the allogeneic knee joints of the SD rat choke vessels that had not been frozen, and the transplantation of the allogeneic knee joints of choke vessels plus the measurement of the contents of IL-2, IFN-γ, IL-4, and IL-10 before and after the transplantation. It tries to further explore the effect of refrigeration on the immunoreaction of the allogeneic osteoarticular joints of choke vessels from the perspective of immunology.Methods: Grouping: Among the 55 SD rats, 15 were taken as the donors of knee joints, and then the first group was the control group, the second group is the one of transplantation of allogeneic osteoarticular joints of choke vessels, the third group is the one of transplantation of cryopreserved allogeneic osteoarticular joints of choke vessels, and the fourth is the one using Cy A for 14 days after the transplantation of allogeneic osteoarticular joints of choke vessels, each of which consisted of ten cases.The animal models of this experiment had referred to the methods designed by Yaremchuk et al to separate and retain a long section from the blood vessel of the knee joints of the donors, and in the meanwhile also reserve part of the muscle sleeves, then cut the femoral artery and femoral vein and saw off the femur, tibia and fibular at the inguinal ligament of the receptor SD rats. During the transplantation, the knee joints with blood vessels were planted below the skin of the abdomen of the receptor and the bloodvessels were anastomosed to the ends of the femoral artery and femoral vein of the receptor SD rats. The knee joints cut from the donors for the freezing group were put into the cryopreservation tubes with 15% DMSO cryoprotectant and then stored in a refrigerator at 4℃ for 1.5 hours, after which they were transferred to an ultra-low temperature refrigerator so that the temperature could be reduced to- 60℃ at the rate of 1℃/min, which was maintained for 1 hour. Then they were thrown into liquid nitrogen at the temperature of-196℃ for 30 days of preservation. The knee joints from the donor were taken out from the liquid nitrogen before the transplantation and put into the water at the temperature of 37 ℃ for rewarming, and heparin saline solution was taken to flush the tube so as to eliminate the cryoprotectant. After the operation, 40,000 U /kg.d of penicillin was administered to every group through intramuscular injection for altogether three days plus 0.3g aspirin through intramuscular injection and oral taking of 25 mg dipyridamole dissolved in 250 ml of water were conducted for seven consecutive days. As for the fourth group,cyclosporine(Cy A) was dissolved in 99% dimethyl amide(DMF) so as to render the concentration to reach 10mg/ml, which was administered through intramuscular injection at 5mg/kg.d since the date of the operation and for 14 consecutive days. The peripheral blood of SD rats in all groups were drawn before the operation and one week, two weeks, three weeks,four weeks, six weeks, eight weeks, ten weeks, and twelve weeks after the operation. In addition, flow cytometer method was adopted to examine the number of T cells in the plasma and the changes of the CD4+/CD8+ and CD25+ cells. ELISA method was taken to measure IL-2,IFN-γ,IL-4,and IL-10. The double antibody sandwich method. T test was adopted to conduct comparison on the data in different groups.Results: The two groups after CD25 +,CD4 + / CD8 +, IL-2, IFN-γ, IL-4, IL-10 increased significantly, with the other groups were significantly different,no statistics among the three groups and four groups of both learn thedifference, after CD25 +, CD4 + / CD8 +, IL-2, IFN-γ, IL-4, IL-10 were all increased compared with a statistically significant set of data.Conclusion:Deep frozen for vascularized allograft transplantation and freshly transplanted bone and joint short-term application Cy A reduced immunogenicity is basically the same, to improve graft vascularized allograft bone joints and tissue survival environment.