Establishment Of Improved Vascularized Ilium Transplantation Animal Model

Transplantation is the ideal method to remedy the organ failure and the local tissues which are injured severely. But the rejection cannot be avoided after operation.With the appearing of epochal immunosuppressor,for example cyclosporine A, transplantation faces the new opportunity. But its adverse effects still influence the patients’ life quality.Hence, recent objective is how to relieve or withdraw immunosuppressor.Recently, bone marrow transplantation(BMT) has been mainly used to induce tolerance.Nevertheless,vascularized bone marrow transplantation(VBMT) which derived from composite tissue allotransplantation(CTA),is an unique bone marrow cell transplantation.It contains the bone,the marrow cells, and the bone marrow stromal cells microenvironment.All of them are transplanted in recipient.Compared with the bone marrow infusion,it will produce the donor bone marrow cells continuously. In addition, it can avoid myeloablative, and seldom occur graft-versus-host disease(GVHD).Recent study shows that VBMT can establish the chimerism and have the potency to induce the immune tolerance.Most VBMT animal model are the femurs,however, in the view of clinical transformation,ilium is more likely to be applied to the clinic.Hence, improving and establishing a more feasible animal model of vascularized iliac bone marrow transplantation has the significant value in use.Objective:(1)To explore a relatively simple and stable way of operation of vascularized iliac bone marrow transplantation and establish a reliable animal model for it which can establish the chimerism and induce the immune tolerance.(2)After operation, detecting the chimerism. Using Micro-CT to scan the transplant area and perform the three-dimensional reconstruction,then judging the changes of transplanted iliac bone mineral density. Dynamically monitoring the changes of the ratio of CD4+/CD8+ T cells and the levels of serum Interleukin-2( IL-2) in peripheral blood.Then compared with normal rats and judge the immune status of rats which establish the chimerism after operation.Methods:Selecting male Sprague Dawley(SD) rats as donors and female SD rats as recipients,then performing the modified operation.Donor’s gluteus maximus was striped, and the donor’s iliac which jointed muscle and skin flap was transplanted in the recipient’s groin area.The common iliac artery and iliolumbar vein of the donor pedicle were anastomosed with the femoral artery and vein of the recipient.The experiment was divided into three groups: normal rats group;improved operation group;traditional operation group, namely, without stripping the muscle and the donor’s aorta abdominalis was anastomosed with recipient’s femoral artery.Offering the ciclosporin A after the transplantation(2) Selecting male SD rats as donors, female SD rats as recipients, then performing the modified operation and offering the ciclosporin A after the transplantation.The experiment was divided into three groups:positive group,namely,male SD;negative group,namely,female SD rats,experiment group,namely,improved operation.Drawing blood on the day 28,and extracting the haemal RNA. Then converting it into cDNA by R-T PCR.Detecting rat SRY gene by PCR.Using Micro-CT to scan the transplant area and perform the three-dimensional reconstruction.Dynamically monitoring the changes of the ratio of CD4+/CD8+ T cells by flow cytometry after operation. Dynamically monitoring the changes of the levels of serum IL-2 by enzyme linked immunosorbent assay(Elasa)after operation.Results:(1)Successfully establishing a feasible and improved vascularized iliac bone marrow transplantation.The durations of arterial anastomosis was 24.7±2.3 min.Venous anastomosis time was 33.0±1.6 min.Ischemia time was 102±3.5 min.(2) Successfully detecting the SRY gene in female rats which received the male rats’ iliac bone transplantation by PCR.After Micro-CT scanning and three-dimensional reconstruction,it showed that the transplanted iliac bone mineral density was decreased.(3)Comparing rats which established the chimerism with normal rats,the ratio of CD4+/CD8+ T cells and the levels of serum IL-2 had no difference.Conclutions:(1)From stripping the gluteus maximus and changing the artery anastomosis vessels,we could set up a relatively simple and stable vascularized iliac bone marrow transplantation model.(2)The transplanted iliac bone mineral density was decreased.(3)Through the immunosuppressant,the immune status of the rat after operation was well.On the basis of no rejection,we successfully established an improved vascularized iliac bone marrow transplantation model which could establish the chimerism.