miR-20b对Th17细胞分化和实验性自身免疫性脑脊髓炎发病的调控机制研究

发布时间：2018-06-20 01:42

本文选题：microRNA + Th17　；参考：《南开大学》2014年博士论文

【摘要】：Th17细胞作为第三类效应性CD4+T细胞,在多发性硬化症(multiple sclerosis, MS)中发挥重要的致病作用。实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是一种针对人类MS建立的被广泛应用的动物模型。EAE是由CD4+T细胞介导的中枢神经炎症,组织学表现为中枢神经脱髓鞘及炎症性Thl和Th2细胞浸润。miRNA是一类小的非转录RNA,通常通过转录后调控影响靶基因表达。目前,大量研究表明miRNAs在Thl7细胞诱导的自身免疫疾病方面发挥重要作用,例如人类的MS。有研究显示miR-20b在MS临床病人的外周血细胞中低表达,但是miR-20b在Th17细胞分化及EAE中的调控作用未见报道。我们首先建立了EAE,作为探讨miR-20b对于MS致病作用影响的动物模型。我们发现miR-20b在来自于EAE小鼠的CD4+T细胞中显著低表达。此外,通过将naive CD4+T细胞在体外以natural、Th1、Th2、Th17、Treg不同方向分化而检测其中miR-20b的表达,我们发现相对于natural CD4+T细胞,miR-20b在Th17细胞中显著低表达,而在Th1、Th2和iTreg细胞中则变现为表达上升。基于上述结果,我们推测niR-20b可能通过影响Th17分化而在EAE的发病过程中参与调控。因为miRNAs主要通过其靶基因发挥调控作用,因此我们继续寻找与Th17细胞分化相关的miR-20b可能的靶基因。我们利用小鼠TargetScan数据库来预测miR-20b的备选靶基因。通过荧光素酶检测、GFP抑制实验和免疫印迹实验,我们鉴定并确定RORyt和STAT3为miR-20b可能的靶基因。我们还发现miR-20b分别在mRNA水平抑制STAT3基因表达,在转录后水平抑制RORyt基因表达两种不同机制发挥其调控作用。考虑到RORγt和STAT3作为Thl7细胞分化的主要调控基因,我们推测miR-20b可能直接参与Th17细胞分化的调控。在Th17细胞体外分化过程中,通过分别转染miR-20b的阴性对照(NC)、抑制序列(inhibitor)及模拟序列(mimics),我们发现mimics转染组IL-17A表达显著下调,而inhibitor转染组IL-17A表达轻微上调。相同的,三组IL-17F表达与IL-17A表达类似。与此一致,我们发现在三组转染细胞的培养上清中,IL-17A和IL-17F的表达也可以被miR-20b的mimics显著下调。我们进一步将miR-20b NC. inhibitor及mimics用FAM荧光标记,以检测转染阳性细胞中Th17细胞相关IL-17A和IL-17F的表达,结果与上面类似。上述结果证明miR-20b可以在体外负向调控Th17细胞的分化。为了进一步验证我们的预测,即niR-20b可能在体内调控EAE的发病进程,我们构建了pri-miR-20b慢病毒表达载体。小鼠尾静脉分别注射表达pri-miR-20b (LV-miR-20b)及阴性对照(LV-NC)的慢病毒,7天后,诱导EAE发病,结果显示LV-miR-20b感染组小鼠发病分值统计显著减轻。腰髓切片组织学分析显示]LV-miR-20b感染组的小鼠炎症浸润及脱髓鞘情况也显著缓解。此外,小鼠免疫后14天尾静脉注射上面两种慢病毒,结果显示LV-miR-20b感染组的小鼠疾病严重程度亦有所下降,这些结果提示miR-20b能够抑制EAE的发病进程。为继续深入探讨miR-20b是否能够在EAE发病过程中对于Th17细胞的产生有所影响。我们收集LV-miR-20b及LV-NC感染小鼠中枢神经系统的单核细胞进行分析,结果显示在LV-miR-20b感染组小鼠的中枢神经系统中CD4+T细胞浸润显著减少,进一步胞内细胞因子分析结果显示Thl7细胞比例也显著降低。在LV-miR-20b感染组小鼠的引流淋巴结中,我们也发现更少的Th17细胞浸润,此外,将这些淋巴细胞体外培养并用诱导EAE发病的MOG35-55再刺激,LV-miR-20b感染组的细胞培养上清中IL-17A的表达也相应减少。上述结果表明miR-20b在EAE发病过程中可以在体内抑制Th17细胞的产生。最后,我们发现EAE发病小鼠脾脏中CD4+T细胞的RORyt和STAT3蛋白表达较正常小鼠显著上升。此外,在都诱导小鼠EAE发病的前体下,相对于LV-NC感染组,LV-miR-20b感染组小鼠脾脏中CD4+T细胞的RORγt和STAT3蛋白表达显著被下调。我们进一步证明miR-20b在体内可能通过靶向调控RORyt和STAT3表达来抑制EAE的病程发展及Th17细胞产生。总之,我们的结果展示了1niR-20b在Th17细胞分化以及EAE发病过程中的重要调控作用,基于此,miR-20b可能成为人类多发性硬化症的诊断生物标记或是治疗靶点。
[Abstract]:Th17 cells, as third types of effector CD4+T cells, play an important role in the pathogenesis of multiple sclerosis (MS). Experimental autoimmune encephalomyelitis (Experimental autoimmune encephalomyelitis, EAE) is a widely used animal model for human MS, which is mediated by CD4+T cells. Central neuroinflammation, histologically characterized by demyelination of the central nervous system and inflammatory Thl and Th2 cells infiltrating.MiRNA is a small non transcriptional RNA which usually affects target gene expression through post transcriptional regulation. At present, a large number of studies have shown that miRNAs plays an important role in the autoimmune disease induced by Thl7 cells, such as the study of human MS.. The expression of miR-20b was low in peripheral blood cells of MS patients, but the regulation of miR-20b on Th17 cell differentiation and EAE was not reported.
We first established EAE as an animal model to explore the effect of miR-20b on the pathogenicity of MS. We found that miR-20b was significantly lower in CD4+T cells from EAE mice. In addition, we detected the expression of naive CD4+T cells in the different directions of natural, Th1, Th2, Th17, and Th17. We found the relative expression of naive CD4+T cells in different directions. In natural CD4+T cells, miR-20b is significantly lower in Th17 cells, while in Th1, Th2 and iTreg cells, the expression rises. Based on the above results, we speculate that niR-20b may be involved in the regulation of EAE in the pathogenesis of EAE, because miRNAs mainly through its target gene, we continue to seek. Find miR-20b target genes related to Th17 cell differentiation.
We used the mouse TargetScan database to predict the candidate gene for miR-20b. Through luciferase detection, GFP inhibition and Western blotting, we identified and identified RORyt and STAT3 as the possible target genes for miR-20b. We also found that miR-20b inhibits the STAT3 gene expression at the mRNA level and inhibits the RORyt gene at the post transcriptional level. Two different mechanisms are expressed to play its regulatory role.
Considering that ROR gamma T and STAT3 are the main regulatory genes of Thl7 cell differentiation, we speculate that miR-20b may be directly involved in the regulation of Th17 cell differentiation. During the differentiation of Th17 cells, the negative control (NC), inhibition sequence (inhibitor) and the quasi sequence (mimics) of miR-20b, respectively, are found in the Th17 cell differentiation process. The expression of IL-17A in the inhibitor transfected group was slightly up. The same, three groups of IL-17F expressions were similar to IL-17A expression. We found that in the culture supernatant of the three transfected cells, the expression of IL-17A and IL-17F could also be significantly downregulated by miR-20b mimics. The results showed that the expression of Th17 cells related to IL-17A and IL-17F in the transfected positive cells was similar to that above. The results showed that miR-20b could negatively regulate the differentiation of Th17 cells in vitro.
To further verify our prediction that niR-20b may regulate the pathogenesis of EAE in the body, we constructed the pri-miR-20b lentivirus expression vector. The tail veins of mice were injected with the lentivirus expressing pri-miR-20b (LV-miR-20b) and negative control (LV-NC) respectively. After 7 days, the pathogenesis of EAE was induced. The results showed the incidence of LV-miR-20b infection in mice. The histological analysis of the lumbar spinal section showed that the inflammatory infiltration and demyelination of the mice in the]LV-miR-20b infection group were also significantly relieved. In addition, the mice were injected with two kinds of lentiviruses at the tail vein 14 days after immunization. The results showed that the severity of disease in the LV-miR-20b infected mice was also decreased. These results suggest that miR-20b can be used. Inhibition of the progression of EAE.
To further explore whether miR-20b could affect the production of Th17 cells during the pathogenesis of EAE. We collected mononuclear cells from the central nervous system of LV-miR-20b and LV-NC infected mice. The results showed that the infiltration of CD4+T cells in the central nervous system of the LV-miR-20b infected mice was significantly reduced and further intracellular fine. The results of cell factor analysis showed that the proportion of Thl7 cells also decreased significantly. In the drainage lymph nodes of the LV-miR-20b infected mice, we also found less Th17 cell infiltration. In addition, the lymphocytes were cultured in vitro and stimulated with MOG35-55 induced by EAE, and the expression of IL-17A in the cell culture supernatant of the LV-miR-20b sensitive group was also corresponding. These results suggest that miR-20b can inhibit the production of Th17 cells in vivo in the pathogenesis of EAE.
Finally, we found that the expression of RORyt and STAT3 protein in CD4+T cells in the spleen of EAE mice was significantly higher than that in normal mice. In addition, the ROR y t and STAT3 egg white expression of CD4+T cells in the spleen of LV-miR-20b infected mice were significantly lower than those of the LV-NC infected group. In vivo, it may inhibit the progression of EAE and the production of Th17 cells by targeting the regulation of RORyt and STAT3 expression.
In conclusion, our results show the important regulatory role of 1niR-20b in the differentiation of Th17 cells and the pathogenesis of EAE. Based on this, miR-20b may be a diagnostic biomarker of human multiple sclerosis or a target for treatment.
【学位授予单位】：南开大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R744.51

【共引文献】