The Function Of Kupffer Cell In The Tolerance Of Orthotopic Liver Transplantation In Rats

Objective: 1 .To build the model of orthotopic liver transplantation(OLT) in rats,the Wistar to SD rats group were to observe the acute rejection after the transplant, SD to SD were to observe the immune tolerance, we also build a third group by pretreatment of Wistar rats with Gadolinium chloride(Gdcl3) ,which can block phagocytosis of Kupffer cells(KC) of the donor liver. 2.Detecting the cell adhesion molecules(CAMs) and CD 14 expresses and lymphocytes apoptosis in the grafts tissues, measuring the levels of TNF- a , TGF- β , IL-10 in the plasm of recipient rats,liver functions is also measured. 3.Isolating the KC from the grafts,isolating spleen lymphocytes from the SD rats, co-culture the KC and lymphocytes, then,detect the proliferate rates of the lymphocytes. By all these experiments,we want to elucidate the functions of KC in the induction of tolerance of liver transplantation in rats.Methods: l.Orthotopic liver transplantation in rats was performed by the "two cuffs" method.The rats were randomly divided into 3 groups:group A, Wistar->SD to observe the acute rejection;group B, SD->SD to observe theimmune tolerancejand group C,pretreatment of Wistar rats with Gdcl3(10mg/ml) via the tail vein 24h before liver harvesting.5 or 7 days after the operation,recipient rats were killed,and samples were collected.Recipients survival rate ,grafts histopathological and ultrastructural characteristics were detected,plasm levels of alanine aminotransferases (ALT),total bilirubin(TB),albumin(ALB) were measured with an automatic biochemical analyser.Expression of intercellular adhension molecules- l(ICAM-l), p-selectin,e-selectin,and integrin- a L(LFA-1),CD14 were detected using immunohistochemical staining.Plasm levels of TNF-a,TGF-P,IL-10 were measured with enzyme linked immunosorbent assay(ELISA).apoptosis of the infiltrating lyphocytes were detected by TUNEL method. 2.KCs were isolated 5 or 7 days after the OLT by a modified two-step perfusion method. Spleen lymphocytes were isolated from a non-operated SD rats.Co-culture the KC and leukocytes for 72 hours,then measure the proliferation of leukocytes using MTT method,CD4+,CD8+ were measured by flow cytomery(FCM).Results: 1.Liver transplantation model of SD rats to recipient SD rats can result in tolerance of liver graft and prolonged survival of recipient rats.The survival rate of this group after 1 week was 100%.histological examination showed that mild morphologic changes occurred in grafts tissue.mild necrosis of hepatocytes were found,hepatic sinusoid expanded slightly and filled with a few erythrocytes,sinusoidal endothelial cells(SEC) were slightly swollen,but the structure of hepatic lobules remained.there was little lymphocytes infiltrating the portal tracts region.There was slight increase in liver enzyme activity without significant changes of TB and ALB levels.theexpression of CAMs and CD 14 is lower than the other two groups,but the apoptosis rates of the infiltrating lymphocytes and the levels of TGF- P and IL-10 in plasm is higher than them, but the TNF- a level is lower than them too.KC isolated from this group's recipients have little effects on the proliferation of SD spleen lymphocytes. 2.Liver transplantation of Wistar rats to recipient SD rats can results in acute rejecting activity,survival rate of recipient rats after 1 week was 60%,histological examination showed servere tissue injury.The marked pathological features indued remarkably wider sinusoidal spaces, erythrocytes filled the sinusoidal spaces.hepatocytes and sinusoidal SEC were swollen,severely shrunked and necrotic hepatocytes could be observed.bile capillaries were narrow and some of them were destroyed.there were severe lymphoctes infiltrating.Judged by significant increase of ALT and TB levels and apparent decrease of ALB level,the liver function was severely impaired.The expression of CAMs and the CD 14 in the grafts and the TNF-a level of the recipient plasm is higher than the group B, but the TGF- P , IL-10 level is lower than group B,so is the apoptosis rates of the infiltrating lymphocytes. KC isolated from this group can stimulate the proliferation of SD spleen lymphocytes more actively than the group B. 3.In group c,after 5 or 7 days of the OLT,the grafts showed a same morphologic changes like group A,but the degree is more severe.the expression of CAMs and CD 14 in the grafts like the group A,is higher than the group B, but the TNF- a of this group's recipients is the highest.on the other side,it's TGF- {3 JL-10 is the lowest.KC isolated from this group have a active effect on the SD spleen lymphocytes proliferation,especially the CD8+.Conclusion : 1 .Liver transplantation from donor Wistar rats to recipient SD rats can result in acute rejection activity ,and liver transplantation of donor SD to recipient SD is a toleranced model. 2.Kupffer cells play a active role in the induce of acute rejection in liver transplantation by upregulation of TNF~ a secretion, it is also play an important role in the mechanism of liver transplatntion acceptance by the release of IL-10,TGF-p.Gdcl3 can block KC's immunosuppressive effect by inhibit the release of IL-10,TGF-pof KC.