| 【Background】Liver transplantation is the only effective mean to cure for end-stage liver diseases, while the grafts will face the immune rejection and loss the function. To induce the immune tolerance, life-long immune inhibitors bring high infection rate, high tumor occurrence and recurrence rate, and heavy economic burden. How to reduce and avoid the use of immunosuppressants is a hot issue in the study of liver transplantation [1]. Cytotherapy with immunoregulatory cells is a potential way [2]. The im DC and Treg cells could regulate the donor’s immune system.Current studies indicated that im DC and Treg interact with each other in the regulation of immune. Im DC is known to promote Treg differentiation and proliferation of Treg, while Treg can maintain the undifferentiated state of im DC. But overall, the im DC play a central role [3]. However, the former is restricted to its short storage time, and can be easily activated into mature state. Fortunately, DC could secrete membrane vehicles--Exosome, which is rich in immune related molecules in its surface, and contains DC proposed exogenous antigen presenting, even with a certain amount of genetic material. The exosome has good stability, and can be preserved for long time under low temperature [4]. In order to solve the problem of restrictions and weak effect of tolerance induction, previous research using donor im DC derived exosome(im Dex) to protect grafts. The results showed that im Dex can prolong the survival time of the donor heart and small intestine graft rodents, and the protective effect in vivo has close relationship with the proportion of Treg [5, 6]. In addition, to induce the ideal state of tolerance, small dose of immunosuppressive drugs are needed to be combined with. We assumed that, similar to im DC, im Dex is also through the amplification of Treg quantity to enhance the function of immune regulation in vivo. 【Aims】In order to further reduce the dose of immunosuppressive drugs, and verify our hypothesis. We constructed inbred rats orthotopic liver transplantation model. With the donor derived antigen-specific im Dex and Treg, we observed protective effects of the combined therapy, and explore the interaction between im Dex and Treg both in vivo and in vitro. 【Methods】Use BN/F344 rats as donor, Lewis rats as receptor, to establish orthotopic liver transplantation model.To obtain donor derived im Dex, ultra high speed gradient centrifugation was used. Electron microscopy and flow cytometry were used to identify the phenotype of im Dex.Different doses of im Dex treatment were studied, and survival analysis was performed to explore the protective effect of plant moved to liver, and investigate the relationship between the effect and the dose.Using the two steps magnetic isolation method to get CD4+ CD25+ T cells from the spleen of recipients, and then co-culture them with donor antigen presenting cells to obtain donor antigen pre-stimulated Treg. The change of their functional molecules was identified with Flow cytometry, and immunosuppressive effects were also observed both in vivo and in vitro.Different source and different specificity of Treg were used for transplantation to verify the importance of antigen specificity of Treg in transplantation immunity.Furthermore, in vivo experiments, im Dex along with Treg were used to observe possible protective effect on graft and changes in immune status with survival analysis and mixed lymphocyte reaction assay.In vitro, experiments were used to explore the effect of im Dex on Treg. Treg cells labelled with CFSE were infused into recipient, to observe the relationship between im Dex and Treg, and verify the results obtained above. 【Results】1) Im Dex obtained with ultra-high speed gradient centrifugation maintained the classic phenotype of im DC, the optimized dose is 20 μg.2) Protective effects of donor antigen specific Treg was significantly higher than that of natural Treg sorted by magnetic activated cell sorting(P<0.05).3) The survival time of the group treated with combination use of im Dex and Treg, was longer than the control group(P<0.01); Immunological rejection was slight in combination therapy group both at the 10 th day and 40 th day; T activity of cell proliferation and cytokine secretion were reduced. Exogenous Treg cells proliferated significantly in vivo, and was distributed into graft, spleen and the nodes of the recipients.4) Further in vitro experiments found that im Dex could stimulate the proliferation of Treg, and proliferation rates peaked at the 5th day in the high dose group, but proliferation rates were similar among different doses groups at the 7th, which suggested that high dose of im Dex may not be required. This result was similar with in vivo experiments mentioned above.5) In vivo experiments indicated that, the proliferation of Treg in co-treated group was significantly higher than that of exogenous Treg treated along group, which was consist with the results in vitro. 【Conclusions】Im Dex can be stored under low temperature for more than half a year, which solve the limitation of preservation of im DC, and can be applied to clinical transplantation to induce immune tolerance state; we found that the combined therapy with im Dex and Treg can achieve stable immune tolerance state and avoid the adverse reaction caused by immunosuppressive drugs. This combined therapy can be expected to provide a feasible scheme for clinical inducing transplantation immune tolerance. |
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