發(fā)布時(shí)間：2018-06-20 01:42

  本文選題：microRNA + Th17�。� 參考：《南開大學(xué)》2014年博士論文

【摘要】：Th17細(xì)胞作為第三類效應(yīng)性CD4+T細(xì)胞,在多發(fā)性硬化癥(multiple sclerosis, MS)中發(fā)揮重要的致病作用。實(shí)驗(yàn)性自身免疫性腦脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是一種針對(duì)人類MS建立的被廣泛應(yīng)用的動(dòng)物模型。EAE是由CD4+T細(xì)胞介導(dǎo)的中樞神經(jīng)炎癥,組織學(xué)表現(xiàn)為中樞神經(jīng)脫髓鞘及炎癥性Thl和Th2細(xì)胞浸潤。miRNA是一類小的非轉(zhuǎn)錄RNA,通常通過轉(zhuǎn)錄后調(diào)控影響靶基因表達(dá)。目前,大量研究表明miRNAs在Thl7細(xì)胞誘導(dǎo)的自身免疫疾病方面發(fā)揮重要作用,例如人類的MS。有研究顯示miR-20b在MS臨床病人的外周血細(xì)胞中低表達(dá),但是miR-20b在Th17細(xì)胞分化及EAE中的調(diào)控作用未見報(bào)道。 我們首先建立了EAE,作為探討miR-20b對(duì)于MS致病作用影響的動(dòng)物模型。我們發(fā)現(xiàn)miR-20b在來自于EAE小鼠的CD4+T細(xì)胞中顯著低表達(dá)。此外,通過將naive CD4+T細(xì)胞在體外以natural、Th1、Th2、Th17、Treg不同方向分化而檢測其中miR-20b的表達(dá),我們發(fā)現(xiàn)相對(duì)于natural CD4+T細(xì)胞,miR-20b在Th17細(xì)胞中顯著低表達(dá),而在Th1、Th2和iTreg細(xì)胞中則變現(xiàn)為表達(dá)上升。基于上述結(jié)果,我們推測niR-20b可能通過影響Th17分化而在EAE的發(fā)病過程中參與調(diào)控。因?yàn)閙iRNAs主要通過其靶基因發(fā)揮調(diào)控作用,因此我們繼續(xù)尋找與Th17細(xì)胞分化相關(guān)的miR-20b可能的靶基因。 我們利用小鼠TargetScan數(shù)據(jù)庫來預(yù)測miR-20b的備選靶基因。通過熒光素酶檢測、GFP抑制實(shí)驗(yàn)和免疫印跡實(shí)驗(yàn),我們鑒定并確定RORyt和STAT3為miR-20b可能的靶基因。我們還發(fā)現(xiàn)miR-20b分別在mRNA水平抑制STAT3基因表達(dá),在轉(zhuǎn)錄后水平抑制RORyt基因表達(dá)兩種不同機(jī)制發(fā)揮其調(diào)控作用。 考慮到RORγt和STAT3作為Thl7細(xì)胞分化的主要調(diào)控基因,我們推測miR-20b可能直接參與Th17細(xì)胞分化的調(diào)控。在Th17細(xì)胞體外分化過程中,通過分別轉(zhuǎn)染miR-20b的陰性對(duì)照(NC)、抑制序列(inhibitor)及模擬序列(mimics),我們發(fā)現(xiàn)mimics轉(zhuǎn)染組IL-17A表達(dá)顯著下調(diào),而inhibitor轉(zhuǎn)染組IL-17A表達(dá)輕微上調(diào)。相同的,三組IL-17F表達(dá)與IL-17A表達(dá)類似。與此一致,我們發(fā)現(xiàn)在三組轉(zhuǎn)染細(xì)胞的培養(yǎng)上清中,IL-17A和IL-17F的表達(dá)也可以被miR-20b的mimics顯著下調(diào)。我們進(jìn)一步將miR-20b NC. inhibitor及mimics用FAM熒光標(biāo)記,以檢測轉(zhuǎn)染陽性細(xì)胞中Th17細(xì)胞相關(guān)IL-17A和IL-17F的表達(dá),結(jié)果與上面類似。上述結(jié)果證明miR-20b可以在體外負(fù)向調(diào)控Th17細(xì)胞的分化。 為了進(jìn)一步驗(yàn)證我們的預(yù)測,即niR-20b可能在體內(nèi)調(diào)控EAE的發(fā)病進(jìn)程,我們構(gòu)建了pri-miR-20b慢病毒表達(dá)載體。小鼠尾靜脈分別注射表達(dá)pri-miR-20b (LV-miR-20b)及陰性對(duì)照(LV-NC)的慢病毒,7天后,誘導(dǎo)EAE發(fā)病,結(jié)果顯示LV-miR-20b感染組小鼠發(fā)病分值統(tǒng)計(jì)顯著減輕。腰髓切片組織學(xué)分析顯示]LV-miR-20b感染組的小鼠炎癥浸潤及脫髓鞘情況也顯著緩解。此外,小鼠免疫后14天尾靜脈注射上面兩種慢病毒,結(jié)果顯示LV-miR-20b感染組的小鼠疾病嚴(yán)重程度亦有所下降,這些結(jié)果提示miR-20b能夠抑制EAE的發(fā)病進(jìn)程。 為繼續(xù)深入探討miR-20b是否能夠在EAE發(fā)病過程中對(duì)于Th17細(xì)胞的產(chǎn)生有所影響。我們收集LV-miR-20b及LV-NC感染小鼠中樞神經(jīng)系統(tǒng)的單核細(xì)胞進(jìn)行分析,結(jié)果顯示在LV-miR-20b感染組小鼠的中樞神經(jīng)系統(tǒng)中CD4+T細(xì)胞浸潤顯著減少,進(jìn)一步胞內(nèi)細(xì)胞因子分析結(jié)果顯示Thl7細(xì)胞比例也顯著降低。在LV-miR-20b感染組小鼠的引流淋巴結(jié)中,我們也發(fā)現(xiàn)更少的Th17細(xì)胞浸潤,此外,將這些淋巴細(xì)胞體外培養(yǎng)并用誘導(dǎo)EAE發(fā)病的MOG35-55再刺激,LV-miR-20b感染組的細(xì)胞培養(yǎng)上清中IL-17A的表達(dá)也相應(yīng)減少。上述結(jié)果表明miR-20b在EAE發(fā)病過程中可以在體內(nèi)抑制Th17細(xì)胞的產(chǎn)生。 最后,我們發(fā)現(xiàn)EAE發(fā)病小鼠脾臟中CD4+T細(xì)胞的RORyt和STAT3蛋白表達(dá)較正常小鼠顯著上升。此外,在都誘導(dǎo)小鼠EAE發(fā)病的前體下,相對(duì)于LV-NC感染組,LV-miR-20b感染組小鼠脾臟中CD4+T細(xì)胞的RORγt和STAT3蛋白表達(dá)顯著被下調(diào)。我們進(jìn)一步證明miR-20b在體內(nèi)可能通過靶向調(diào)控RORyt和STAT3表達(dá)來抑制EAE的病程發(fā)展及Th17細(xì)胞產(chǎn)生。 總之,我們的結(jié)果展示了1niR-20b在Th17細(xì)胞分化以及EAE發(fā)病過程中的重要調(diào)控作用,基于此,miR-20b可能成為人類多發(fā)性硬化癥的診斷生物標(biāo)記或是治療靶點(diǎn)。
[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.
【學(xué)位授予單位】：南開大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R744.51

【共引文獻(xiàn)】

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