| Background and Objective:In recent years, a large number of studies have shown that Treg cells play an important role in organ allograft rejection, and the immune rejection response can be controlled through the regulation of Treg cells. If we take steps to induce a sufficient number of Treg cells or enhance their function in vivo prior to transplant rejection or in the early stage of the rejection, we will likely inhibit the rejection process and achieve immune tolerance. However, the exact mechanism of differentiation and development of Treg cells, as well as its role were still unclear. The reasonable way to induce Treg cells in recipient need to be further studied.With the advances in the study of microRNA(miRNA) in recent years, it was found that miRNAs play an important role in homeostasis and function of Treg cells, and the expression of many genes in Treg cells depends on mi RNAs. A lot of studies further screened out the miRNAs differentially expressed between Treg cells and effector T cells, including miR-146 a, which was related with the immune response. This miRNA was regulated by TLRs signaling pathway which participates in the innate immune response in cell membrane, and a variety of factors, TNF-α and IL-1β, etc, were able to induce the expression of mi R-146 a. It was confirmed in the subsequent studies that miR-146 a could regulate the expression of TRAF6, IRAK1, STAT1 at the post-transcriptional level as a feedback regulation, which could affect the body’s immune response. However, the relationship between miR-146 a and Treg in the immune response of organ transplantation has not been studied.The main purpose of this study was to investigate the the molecular mechanism of mi R-146 a in the regulation of Treg cells and enhance the immunosuppressive function of Treg cells by regulating the expression level of miR-146 a in vivo, thereby establishing a method of inhibiting allograft rejection. Methods:The study was divided into three parts: transfection experiments in vitro, transfect--ion experiments in vivo and heart transplantation model experiments. In the first part, expression levels of miR-146 a were regulated in Treg cells by the way of transfection in vitro to study its effect on Treg cells. In the second part, the levels of miR-146 a were regulated through the dorsal penile vein injection of miR-146 a agomir and antagomir in vivo. Transfection efficiency were evaluated and we further explored the role of miR-146 a in Treg cells in vivo. In the third part, heart transplantation models were established using transfected mice in the second part as recipients to study the role of miR-146 a in Treg cells regulating the mice cardiac allograft immune rejection. Results:1. In vitro, miR-146 a agomir and antagomir could significantly up- and downregulated the expression level of miR-146 a in Treg cells(P<0.01). Compared with the control group, the expression levels of target genes TRAF6, IRAK1, STAT1 mRNA and protein were significantly lower in agomir group(P<0.01), while the expresssion were significantly increased in antagomir group(P<0.01). The expression of CD62 L and Ki67 were significantly increased in antagomir group than the other two groups(P<0.01). In addition, CD4+T cell proliferation index in antagomir group was significantly lower(P<0.05) than the other two groups.2. In vivo, compared with the control group, miR-146 a expression levels of Treg cells were significantly change while mi R-146 a agomir and antagomir transfection dosages were separately 10 nmol and 150nmol(P<0.01), while the miR-146 a expression in CD4+CD25-T cells were not affected using the corresponding dose(P>0.05). The expression level of TRAF6, IRAK1, STAT1 mRNA and protein of Treg cells were significantly lower in agomir group(P<0.01), but increase in antagomir group(P<0.01). T cell subsets(CD3+, CD4+, CD8+) in peripheral blood, spleen, thymus in each group showed no statistically significant difference(P>0.05). Treg cell proportions in peripheral blood and spleen cells in antagomir group were significantly higher than the other two groups(P<0.01), while the proportion of Treg cells in the thymus showed no significant difference(P> 0.05). In mixed lymphocyte culture system, compared with the control group and agomir group, CD3+, CD4+T cell proportions were significantly lower in antagomir group(P<0.05), while the proportion of Treg cells were significantly increased(P<0.01), and the proportion of Th1 cells were also significantly increase(P<0.01). the expression of IFN-γ in the supernatant was also significantly higher in antagomir group(P<0.01), whereas the expression of IL-4, IL-17 showed no significant difference(P>0.05).3. In the heart transplantation model experiments, compared with the control group and αIFN-γ group, the survival time of donors in antagomir+αIFN-γ group was significantly longer(P<0.05), and the acute rejection pathological grading of donor heart was significantly lower(P<0.05). The ratios of CD4+T cells in antagomir group and antagomir+αIFN-γ treatment group were significantly lower compared with the saline control group(P<0.05), while the proportion of Treg cells and the expression levels of Foxp3 were significantly higher(P<0.05). Compared with the control group, the expression of IFN-γ in donor hearts in the 5th day after transplantation were significantly higher in antagomir group(P<0.05), and the protein expression levels of STAT1 and pSTAT1 were also significantly increased(P<0.05). But after the intervention with IFN-γ neutralizing antibodies, pSTAT1 expression levels were significantly lower(P<0.05), whereas the expression level of TRAF6 and IRAK1 in each group showed no significant difference(P>0.05). Conclusions:It is effective to regulate the expression of miR-146 a in Treg cells by the way of intravenous injection of miR-146 a agomir and antagomir through penis vein. In physiological conditions, mi R-146 a can regulate the expression of TRAF6, IRAK1, STAT1 in Treg cells. The proliferation ability and suppression function of Treg cells can be enhanced after down regulating of miR-146 a. In rejection process of mice heart transplantation, miR-146 a is mainly involved in the regulation of Treg cells through IFN-γ/STAT1 signaling pathway and the target gene is STAT1. Althogh miR-146 a can also effectively reduce the proliferation of Treg cells, the inhibition of the secretion of IFN-γ of Th1 is impaired. Collaborative application of IFN-γ neutralizing antibodies can inhibit the proliferation of effector T cell and rejection process, and improve the survival of the donor heart. |
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