The Study Of Induced Transplantation Immune Tolerance By Immunosuppressant And Apoptosis Cells

Backgroud:Organ transplantation is the most effective treatment to end-stage organ disease, but graft rejection is still an obstacle against long-term patient and graft survival,the application of conventional immunosuppressive agents can effectively inhibit acute rejection, but can not solve the fundamental cause of the chronic graft rejection, and long-term application in patients will increase the chance of infection, poisoning, and the incidence of cancer and other diseases, besides,it is still expensive. Thus, the solution of rejection after organ transplantation is to induce the recipient to the donor graft immune tolerance, immune tolerance means the body's immune system is led to non-antigen-specific immune response after exposure to certain antigens, once the formation of such immune unresponsiveness succeeds, it is not necessary to maintain any immunosuppressive agent, so it will solve post-transplant rejection, granting graft and host with a long-term coexistence. However, at present in transplantation immunology area,there is a challenge that how to induce immune tolerance of transplant recipient? Accordingly, Professor Sun erwei proposed his hypothesis of using donor's apoptotic cells to induce receptor's immunological tolerance. Apoptosis is a common physiological and pathological phenomenon in the body, the hypothesis indicates that the apoptotic cells can initiatively regulate the immune function, apoptotic cells send to APC initiative through swallowing the signal transmission of "eat me signal",it makes the phagocytic uptake of apoptotic cells were able to quickly and carried by donor antigens which is specifically concentrated within and recipient APC, thereby providing a large number of donor MHC antigens by recipient APC to effectively presented to T cells in a short time, while uptake of apoptotic cells by APC, the APC to promote secretion of immunosuppressive factors, and to promote regulatory T cells'(Treg) proliferation to induce immune tolerance. Fsdok and others also found that it can inhibit the secretion of several inflammatory factors, and promoted TGF-β, IL-10 and some other immune-regulating factors' production, when a large number of apoptotic cells were phagocyted by macrophage. Voll, who found that in response of LPS stimulated peripheral blood lymphocytes, then by adding apoptotic cells, it not only inhibited the secretion of IL-2, IL-1B, TNF-αand other inflammatory factors,but also boosted the immune inhibitory factor IL-10 generation. The research group has successfully established a pre-infusion of donor apoptotic cells induced small animal's heart, liver transplant tolerance model, but it is not an obvious effect of induction immune tolerance when the simple infusion of apoptotic cells on large animal transplantation model, similarly, many scholars in China and abroad-induced immune tolerance of adult large animal transplantation conducted a number of attempts, but they did not get the ideal outcome, however, the apoptotic cells combined with immunosuppressive drugs induce large animal transplantation immune tolerance has not been reported.Currently how to build an adult large animal model and clinical induction of immune tolerance has become the main direction and objective of transplantation immunology academic. Because the immune system of the big animals is relatively complex,and they have very strong rejection, and it is too expensive, so in this discussion, we established the skin graft model in mice,the mouse transplantation rejection is strong, and the cost of mice is lower, to induce immune tolerance of the recipient mice by pre-infusion of apoptotic cells and combined with low doses of immunosuppressive drugs to explore its mechanisms and principles by detecting cytokines and changes of Treg cells at different time points after transplantation to do an exploratory study for further research on large animals and clinical induction of immune tolerance.The topic is the National Natural Science Foundation of China (those mechanisms for apoptosis inducing transplantation immune tolerance) funded projects. In this study, using the same allogeneic transplantation model as mouse skin transplantation,as we know, a skin graft is the strongest of a viable organ transplant rejection, it has been widely used as transplantation model,because observation period is short, easy to operate, a high success rate,easy to control, and so on; Preparation of apoptotic cells have various methods, at present it is commonly recommended that ultraviolet radiation, antibody induction, chemotherapy drug-induced, radiation-induced, hormone-induced and other methods, this experiment adopts dexamethasone drug-induced method. Dexamethasone as a glucocorticoid drugs,it is capable of regulating cell growth, development and inducing death to achieve the maintenance of body tissue stability. That Dexamethasone-induced lymphocytes and other immune cell apoptosis is more stable and easier to operate have been more reported in the literature than the other method.Part 1 A preliminary study of the dexamethasone induced thymocytes apoptosis of donorPurpose:To explore the effects of dexamethasone induced apoptosis of mouse thymocytes, in order to find a approach of high stability, easy to control, and a high induce rate of apoptosis for further experiments.Method:Mulling the healthy adult mouse thymus cell to be suspension, cell count and then adjusting the cell concentration reach 1×106/ml with RPMI-1640 complete medium (containing 10% fetal calf serum), the cells were divided into two groups:one group with dexamethasone mg incubate in 5%CO237℃incubator for 6 hours; the other exposed to UV lamp which placed 20cm Department direct for 15 minutes. Then stain the two groups of post-thymic cell suspension with Annexin V-PI double staining, flow cytometry analysis of thymocytes apoptosis and necrosis.Result:1,The apoptosis rate of mouse thymocytes which was treated with dexamethasone have a little difference.The thymocytes is treatmented by dexamethasone in the concentration of 1×10-7M, then detect the apoptosis rate by flow cytometry,it is 53.72%, when it is treatmented with dexamethasone in 2×10-5M concentration,the apoptosis rate get to 58.46%,and when the dexamethasone concentration change to 2×10-3M, the apoptosis rate get to 55.72%.2,Mouse thymocytes was treated with UV irradiation(keep 20cm distance for 15 minutes)and then incubate in 5% CO237℃incubator for 4 hours,and the apoptosis rate is 50.29%.Conclusions:The dexamethasone-induced apoptosis in mouse thymocytes is complex, time-consuming, when ultraviolet radiation method is simple and concise. However, dexamethasone-induced apoptosis rate in mouse thymocytes average should be higher than the ultraviolet irradiation, and have a low necrosis rate, the results are stable, easy to control,because we can get the highest thymocytes apoptosis rate by dexamethasone in 2×10-5M concentration,, so the following to the recipient mice will use this method.Part 2 The study of immunosuppressive combine apoptosis inducing immunological tolerance of mouse skin transplantationPurpose Explore mechanism of donor apoptotic cells with immunosuppressive induceing skin graft immune toleranceMethod 1 Preparation of donor apoptotic cells in the thymus:C57 mice obtained by grinding the thymus lymphocytes were also incubated into the incubator for 6 hours after adding dexamethasone,then centrifugate to acquire mouse thymus apoptosis cells, flow cytometry analysis the apoptosis ratio of thymocytes2 The recipient mice infusion donor thymus apoptotic cells:respectively 7,3,1 days before operative infusion of donor apoptotic cells in the recipient mice through penile dorsal vein.3 Establishment of mouse back to back skin transplantation model:the reconciliation of donor mouse back skin with 4-8 suture needle fixed in the recipient mice back, covered with sterile dressing then bandage bandaged.4 Using flow cytometry to detect the Treg cells in blood and the use of liquid chip technique detect whole blood cytokines at different time points.Result 1,Skin survival time of 45 cases autologous skin grafts (surgical control group) is more than 1 month, and survival rate was 95%(43/45)2,Pre-infusion apoptotic cells one,two,three times respectively, the skin survival times are not statistically significant(P> 0.05), but the infusion of apoptotic cells three times,skin grafts survive in mice was significantly extended.3,82 cases of back-back of c57-> Babl/C mice skin graft surgery,18 cases for PBS group, the skin survival time is average 6.8 days,in which there are 20 cases apoptosis group, the skin survival time is average 7.4 days,the other 22 cases is rapamycin combine to apoptosis group,the time is average 10.68 days. Apoptotic cell group compared to the PBS control group by Kaplan-Meier is statistically significant, is not significant difference(P> 0.05),compared to rapamycin group, the PBS group have a statistically significant,apoptosis joint rapamycin group compare to the rapamycin group,the result is not statistically significant(P> 0.05).4,78 cases of Babl/C-> C57 mice back-back skin graft surgery,18 cases for PBS group, the skin survival time is average 6.5 days, in which there are 20 cases apoptosis group, the skin survival time is average 7.3 days, the other 19 cases is rapamycin group,the time is average 10:42 days, the other 21 cases is rapamycin combine to apoptosis group,the time is average 10.52 days. Apoptotic cell group compared to the PBS control group by Kaplan-Meier statistically significant, is not significant difference(P> 0.05),but the apoptosis group has an extended tendency. Apoptosis joint rapamycin group was compared to the rapamycin group,the result is not statistically significant(P> 0.05).Conclusion1 Infused apoptotic cells for 1 or 2 times, there was not significantly longer to mouse skin graft survival time, but the survival time by infusion of apoptotic cells 3 times had an obvious extend tendency;2 There is a little effect of apoptosis induce immunosuppressive synergy in mouse skin graft model;3 Low doses of immunosuppressive drugs can prolong allogeneic skin graft survival time of mice;4 There is inapparent effect of apoptotic cells combined immunosuppressive synergy in mouse skin graft model.Part 3 The mechanism study of different immunosuppressive drugs effecting the secretion of whole blood cytokine.Objective:To study the different immunosuppressive drugs are able to affect different cytokines secretion in blood.Methods:1,Sampling:Whole blood from healthy volunteers which were effected by different immunosuppressive drugs,incubate for 6 hours,then stimulated by lOul 0.15μg/ml phorbol ester (PMA) combined 10μl 2.5μg/ml ionomycin (IONO) for 6 hours, centrifuge (300 G,5 min),assemble supernatant for detect.2,Detect the chang of cytokines with Bio-Plex system,compare to the level of cytokines in different immunosuppressive groups.3,Statistics:statistically anlysis by SPSS10.0,compare the different of experiment and control groups by One Way Anova and LSD,it has statistically significant(P< 0.05).Results:High concentration, the middle concentration of dexamethasone (DEX) and cyclosporin A (CsA) can inhibit the secretion of MCP-1, but tacrolimus (FK506) and mycophenolic acid (MPA) can not effectively inhibition of cytokine secretion.Conclusion:DEX and CsA can inhibit the secretion of MCP-1, while FK506 and MPA has no effect on the secretion of MCP-1.