miR-146a调控Treg细胞抑制小鼠心脏移植免疫排斥的研究

发布时间：2018-06-08 21:51

本文选题：miRNA + 调节T细胞　；参考：《天津医科大学》2015年博士论文

【摘要】：研究背景和目的:近年来,大量研究表明Treg细胞在器官移植免疫排斥反应中起着重要的调控作用,通过对Treg细胞的调控能够控制免疫排斥反应。如果在移植前或者在排斥反应的早期阶段采取措施在受者体内诱导产生足够数量的Treg细胞或增强其抑制功能,将可能会减轻排斥反应,实现对移植器官的免疫耐受。尽管如此,Treg细胞的诱导产生、分化发育以及发挥抑制作用的确切机制仍不明确,有关Treg细胞在器官移植中的合理诱导方式、如何保持其最佳作用也有待于进一步的深入研究。近年来随着对microRNA(miRNA)在免疫学领域的研究深入,人们发现miRNA在Treg细胞的稳态和功能方面起着重要的调控作用,很多基因在Treg细胞内持续性地上调或者下调也是依赖于miRNA的。研究人员还进一步筛选出Treg细胞和效应T细胞中差异表达的miRNA,其中包括与机体免疫应答关系十分密切的miR-146a。这种miRNA是在细胞膜表面由参与固有免疫应答中的TLRs信号通路所调控的,而TLRs配体、TNF-α以及IL-1β等多种因素都能够诱导miR-146a的表达。在后续的研究中证实miR-146a能够在转录后水平调控TRAF6、IRAK1、STAT1三者的表达,进而对免疫信号转导通路起到反馈调节作用,从而影响机体的免疫应答过程。但是在器官移植免疫应答过程中,有关miR-146a和Treg的关系却未见报道。本研究主要目的是探讨miR-146a调控Treg细胞的分子机制,并在体内通过调控miR-146a表达水平来增强Treg细胞的免疫抑制功能,从而建立一种抑制器官移植免疫排斥的新方法。研究方法:本研究分为三个部分:体外转染实验、体内转染实验和心脏移植模型实验。在第一部分,通过体外转染调控Treg细胞中miR-146a的表达水平来研究其对Treg细胞的影响,并探讨其机制。在第二部分,通过阴茎背静脉注射miR-146a agomir和antagomir进行体内转染,评价转染效果并筛选出调控Treg细胞mi R-146a表达的最佳转染方案,并进一步探讨体内转染调控miR-146a的表达对小鼠Treg细胞的影响。在第三部分,以第二部分的转染小鼠作为受体,建立腹腔异位心脏移植模型,探讨调控treg细胞mir-146a的表达对同种异系小鼠心脏移植急性免疫排斥的影响,并探讨其机制。研究结果:1.在体外实验中,mir-146aagomir和antagomir可以分别显著上调和下调treg细胞mir-146a的表达水平(p0.01)。与对照组相比,agomir组traf6、irak1、stat1mrna转录水平及蛋白的表达水平均显著下调(p0.01);antagomir组traf6、irak1、stat1mrna转录水平及蛋白的表达水平显著上调(p0.01)。antagomir组treg细胞的cd62l和ki67的表达水平较对照组和agomir组显著升高(p0.01)。另外,treg细胞的体外抑制功能实验中,antagomir组cd4+t细胞的增殖指数较其他两组也明显降低(p0.05)。2.在体内实验中,与对照组相比,mir-146aagomir和antagomir转染剂量分别为10nmol和150nmol时,脾脏中treg细胞的mir-146a表达水平显著改变(p0.01),而相应剂量下的cd4+cd25-t细胞的mir-146a表达却不受影响(p0.05)。agomir组脾脏中treg细胞traf6、irak1、stat1mrna及蛋白的表达水平显著下调(p0.01),antagomir组三者mrna及蛋白的表达水平显著上调(p0.01)。各组外周血、胸腺及脾脏中t细胞亚群(cd3+、cd4+、cd8+)的比例无显著统计学差异(p0.05)。antagomir组外周血和脾脏中treg细胞比例较对照组和agomir组明显升高(p0.01),而三组胸腺中treg细胞比例却无显著性差异(p0.05)。单向混合淋巴细胞培养实验中,与对照组和agomir组相比,antagomir组cd4+t细胞比例显著降低(p0.05),treg细胞比例显著升高(p0.01),ifn-γ阳性表达的细胞(th1细胞)比例显著升高(p0.01)。antagomir组混合培养体系上清液ifn-γ的表达也显著升高(p0.01),而三组il-4、il-17的表达却无显著性差异(p0.05)。3.在心脏移植模型实验中,与生理盐水对照组、antagomir组和αifn-γ组相比,antagomir+αifn-γ组心脏移植物的存活时间明显延长(p0.05),急性排斥反应病理分级亦显著降低(p0.05)。另外,antagomir组和antagomir+αifn-γ组的cd4+t细胞比例较生理盐水对照组明显降低(p0.05),而treg细胞比例以及foxp3在心脏移植物的表达水平均较其余两组显著增高(p0.05)。与生理盐水对照组比较,antagomir组术后第5天心脏移植物ifn-γ的表达显著增高(p0.05),stat1和pstat1的蛋白表达水平显著上调(p0.05)。antagomir协同ifn-γ中和抗体干预后,pSTAT1的表达水平显著下调(P0.05),而各组TRAF6和IRAK1的表达水平却无显著差异(P0.05)。研究结论:经小鼠阴茎背静脉注射miR-146a agomir和antagomir转染试剂能够分别有效的上调和下调Treg细胞内miR-146a的表达。正常生理状态下,mi R-146a在Treg细胞中能够调控TRAF6、IRAK1、STAT1三个靶基因的表达。下调Treg细胞mi R-146a的表达能够增强Treg细胞的增殖能力和对CD4+T细胞的抑制功能。在小鼠心脏移植免疫排斥过程中,miR-146a主要参与调控Treg细胞的IFN-γ/STAT1信号通路,其靶基因是STAT1。虽然miR-146a下调仍能够有效的诱导受体Treg细胞的增殖,但是Treg细胞抑制Th1分泌IFN-γ的功能受损,若协同应用IFN-γ中和抗体则能够有效抑制免疫排斥,从而改善移植心脏存活状况。
[Abstract]:Research background and purpose: in recent years, a large number of studies have shown that Treg cells play an important regulatory role in the immune rejection of organ transplantation, and can control immune rejection through the regulation of Treg cells. A sufficient number of Treg fines are induced in the recipients before or in the early stages of rejection. In spite of this, the induction of Treg cells, differentiation and development, and the exact mechanism to play a inhibitory role are still unclear. The rational inducement of Treg cells in organ transplantation and how to keep their best effects remain to be made. In recent years, with the in-depth study of microRNA (miRNA) in the field of immunology, people have found that miRNA plays an important role in the regulation of the homeostasis and function of Treg cells. Many genes are continuously up-regulated or downregulated in Treg cells and are also dependent on miRNA. Researchers have further screened the Treg cells and the Treg cells. The differential expression of miRNA in effect T cells, including miR-146a., which is closely related to the immune response of the body, is regulated by the TLRs signaling pathway involved in the intrinsic immune response on the surface of the cell membrane, while a variety of factors such as TLRs ligands, TNF- a, and IL-1 beta can induce the expression of miR-146a. Real miR-146a can regulate the expression of TRAF6, IRAK1, and STAT1 three in the post transcriptional level, and then regulate the immune signal transduction pathway, thus affecting the immune response process. However, the relationship between miR-146a and Treg has not been reported in the process of organ transplantation immune response. The main purpose of this study is to explore miR-146a. The molecular mechanism of Treg cells is regulated and the immunosuppressive function of Treg cells is enhanced by regulating the expression level of miR-146a in the body. A new method of inhibiting immune rejection in organ transplantation is established. The research method is divided into three parts: in vitro transfection experiment, in vivo transfection experiment and heart transplantation model experiment. The expression level of miR-146a in Treg cells was regulated by transfection in vitro, and its effect on Treg cells was studied and its mechanism was discussed. In the second part, miR-146a agomir and antagomir were injected into the dorsal vein of the penis to be transfected in vivo. The transfection effect was evaluated and the optimal transfection scheme to regulate the R-146a expression of Treg MI was selected and further improved. To investigate the effect of the expression of miR-146a on mouse Treg cells in vivo transfection. In the third part, a second part of the transfected mice was used as a receptor to establish a model of heterotopic heart transplantation in the abdominal cavity. The effect of the expression of Treg cell miR-146a on the acute immunization rejection of the allogeneic mice was investigated and its mechanism was discussed. The results were as follows: 1 In vitro, mir-146aagomir and antagomir could significantly up-regulate and downregulate the expression level of miR-146a in Treg cells (P0.01). Compared with the control group, the level of TRAF6, irak1, stat1mrna transcriptional level and protein expression in the agomir group were significantly down (P0.01), antagomir group TRAF6, transcription and protein expression level. The expression level of CD62L and Ki67 in Treg cells in group.Antagomir (P0.01) was significantly higher than that in the control group and agomir group (P0.01). In addition, the proliferation index of antagomir group cd4+t cells was significantly lower than that of the other two groups in the inhibition function experiment of Treg cells (P0.05).2. in vivo, compared with the control group. When the transfection dose of tagomir was 10nmol and 150nmol, the miR-146a expression level of the Treg cells in the spleen was significantly changed (P0.01), while the miR-146a expression of the cd4+cd25-t cells at the corresponding dose was not affected (P0.05).Agomir group Treg cell TRAF6 in the spleen. The expression level of RNA and protein was significantly up-regulated (P0.01). The proportion of T cell subgroups (cd3+, cd4+, cd8+) in peripheral blood, thymus and spleen of each group had no significant difference (P0.05), the proportion of Treg cells in peripheral blood and spleen of.Antagomir group was significantly higher than that of control group and agomir group (P0.01), but there was no significant difference in the proportion of Treg cells in the three groups of thymus. (P0.05) in the unidirectional mixed lymphocyte culture experiment, compared with the control group and the agomir group, the proportion of cd4+t cells in the antagomir group decreased significantly (P0.05), the proportion of Treg cells increased significantly (P0.01), and the proportion of ifn- y positive cells (Th1 cells) increased significantly (P0.01).Antagomir group and the expression of ifn- gamma in the mixed culture system was also significantly increased. 0.01) and there was no significant difference in the expression of IL-4 and IL-17 in the three groups (P0.05).3. in the model experiment of heart transplantation, compared with the saline control group. Compared with the antagomir group and the group of alpha ifn- gamma, the survival time of the cardiac allograft in the antagomir+ alpha ifn- group was significantly prolonged (P0.05), and the pathological grading of the acute rejection was also significantly decreased (P0.05). In addition, antagomir group The proportion of cd4+t cells in the group of antagomir+ alpha ifn- gamma was significantly lower than that in the normal saline control group (P0.05), while the proportion of Treg cells and the expression level of Foxp3 in the heart grafts were significantly higher than those in the other two groups (P0.05). Compared with the saline control group, the expression of ifn- gamma in the fifth days after antagomir group was significantly higher (P0.05), stat. The protein expression level of 1 and pstat1 was significantly up-regulated (P0.05) and the expression level of pSTAT1 was significantly down (P0.05), but there was no significant difference in the expression level of TRAF6 and IRAK1 in each group (P0.05). The conclusion was that the miR-146a agomir and the antagomir transfection reagents could be effective by injecting the dorsal vein of the penis in mice. The expression of miR-146a in Treg cells is up and down. Under normal physiological state, MI R-146a can regulate the expression of three target genes of TRAF6, IRAK1, STAT1 in Treg cells. Down regulation of Treg cell mi R-146a can enhance the proliferation of Treg cells and inhibit the inhibitory function of the cells. R-146a mainly participates in the IFN- gamma /STAT1 signaling pathway regulating Treg cells, the target gene is STAT1. although the miR-146a downregulation can effectively induce the proliferation of receptor Treg cells, but Treg cells inhibit the function of Th1 secreting IFN- gamma, and the synergistic application of IFN- gamma neutralization antibody can effectively inhibit immune rejection, thus improving the transplanted heart. Survival.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R654.2

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