| BackgroundIn decades,inducing transplantation immune tolerance,a more attractive method of inducing graft acceptance through the recipient immune response into accepting the transplanted organ,has been the hot point in region of conquering immunology rejection after allograft or exnograft transplantation. The induction and maintenance of immune tolerance to transplanted tissues constitute an active process involving multiple mechanisms that work cooperatively to prevent graft rejection. These mechanisms are similar to inherent tolerance toward self antigens and have a requirement for active immunoregulation that promotes specific unresponsiveness to donor alloantigens.These mechanisms include T cell depletion through activation-induced cell death, "ignorance" of self antigens and the induction of T cell anergy.While these mechanisms are clearly important in the maintenance of self tolerance, they are by themselves not sufficient, as there is also a need for active suppression of autoreactive T cells by Tregs Although initial characterization of these Treg subsets defined their role in the maintenance of tolerance to self, it is now clear that CD4+CD25+Treg cells play an important role in suppressing immune responses directed against alloantigens expressed on transplanted organs and tissues.CD4+CD25+Treg cells have the potent suppressive effect not only on responder T cells either directly via cell contract or secretion of IL-10 and TGF-β1 or alternatively by influencing the stimulating APC but also on B cell Ig response directly via cell contact. Accordingly, CD4+CD25+Treg cells had been look on as effctive tool cells in region of transplantation and autoimmune diseases recently.So it is very meaningful to study the functions of CD4+CD25+Treg cells in vivo.Objective 1. To establish the chronic rejection animal models of kidney transplantation in rats according to the conference,and even to find new ways to enhance the achievement ratio of operation and prolong the survival rate of models.2. To achieve the donor antigenic specificity CD4+CD25+Treg cells in vitro.3. To investigate the dose-effect relationship of donor antigenic specificity CD4+CD25+Treg cells on survival of rat kidney allograft.4. To study the effects of donator antigenic specificity CD4+CD25+Treg cells on the inducing immune tolerance of transplanted kidney in rats.Methods1,Allograft kidney transplantation animal models were established with SD rats as donors ,while Wister rats as recepters.2,CD4+CD25+ T cells were separated from Wister rats'spleens by way of MACS and induced phenotype of donor antigenic specificity in vitro ;3,According to the quantities of prepared CD4+CD25+ T cells inject through tail vein in kidney transplantation , models were devided into four experiment groups : 2X105(groupI),5X105(groupII),1X106(groupIII),2X106(groupIV)(ni=12).The models which had no injection as controls(n=12);Analyses the survival condition of transplanted kidney after kidney transplantation;The level of blood serum creatinine were checked and transplanted kidney histopathology were undertaken at day 4,day 9 and day 15.The results of histopathology were evaluated according to the standard of Banff Schema and gain semi-quantitative scores by way of Watanabe.The reaction indexes of receptor spleen cells to the donor antigens were checken by way of MTT at day 15.4,The treatment group(n=12) moulds were injected CD4+CD25+Treg cells through tail vein at 2,4,6,8,10 weeks after allograft kidney transplantation ,while the controls(n=12) without injection.To observe the mean survival time .To compare the reaction of receptor spleen cells to the donor antigens by way of MTT on day 10, day 40, day 80 postoperative .To detect the level of blood serum creatinine(Cr) on day 10,day 20,day 40,day 60 and day 80 postoperative.Results 1,The achievement ratio of building allograft kidney transplantation models was 91.7%,while it was 77.8% in harvesting chronic rejection models.The mean survival time of chronic rejection models was (66.22±11.89)d.2,CD4+CD25+ Regulatory T Cell( Treg) can be successfully classified by the MACS and the average classifying purity could reach 79.2%±2.8% and the activity of classified cell could be higher than 90 %. There are significant difference in the inhibited rate(IR) between the experimental group I and group II. and we could tell the CD4+CD25+Treg cells isolated owed the phenotype of donor antigenic specificity in vitro.3,The mean survival time of transplanted kidneys was the highest in group 1X106[(31.4±4.6)d]and lowest in controls[(11.7±6.2)d]. Significant diviation was found between groupIII and controls in level of serum creatinine(P<0.05)at day 4,day 9 and day 15; Significant diviation was found between controls with groupIII(P<0.05)in semi-quantitative scores of histopathology.The reaction index of models'spleen cells co-cultivate with donor antigen had significant diviation between controls with groupIIIand groupIV(P<0.05).4,Compared with that in the control,the survival time of transplanted kidney in treatment group was prolonged significantly (P<0.05).There was a significant difference between treatment group with controls in responsing of receptors'spleen cells to donor antigen on day 40 and 80 postoperative (P<0.05). In controls,the level of Cr was significant higher than treatment group on day 20, day 40, day 60 and day 80 postoperative(P<0.05).Conclusion1,Since the slight and the typical pathologic evdience of chronic rejection had been harvested in 8 and 10 weeks after kidney transplantation,can we confirm the sucesses of construction allograft chronic rejection animal modles.2,It is the optimized method for separating the CD4+CD25+Treg cells by way of MACS,and the tool cells could achieved donor antigenic specificity in vitro.3,It can be tentative confirmation that low quantities of CD4+CD25+Treg cells can not prevent the early rejection effectively while the high amount of CD4+CD25+Treg cells will lead to high risk of infection. 1X106 should be the right single dosage in vivo.The tool cells should be injected every two weeks.4,Donor antigenic specificity CD4+CD25+Treg cells can specifically suppress recipient immune response to donor antigen and prolong the survival of transplanted kidney significantly. |
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