miR-146a通过TRAF6调控中枢内源性IFN-β参与实验性自身免疫性脑脊髓炎病程发生发展的机制研究

发布时间：2018-06-16 02:12

本文选题：多发性硬化 + 实验性自身免疫性脑脊髓炎　；参考：《吉林大学》2016年博士论文

【摘要】：实验性自身免疫性脑脊髓炎(EAE)是多发性硬化(MS)的动物模型,是由Th1和Th17细胞介导的一种中枢神经系统自身免疫性炎性脱髓鞘疾病动物模型。在中枢神经系统,渗入的自身活化的Th1/Th17(CD4+T)细胞,CD8+T细胞,巨噬细胞/树突状细胞,肥大细胞以及活化的小胶质细胞,会产生大量的促炎性细胞因子IFN-γ,IL-17,TNF-α,GM-CSF和IL-23等,促进细胞介导的免疫反应。尽管发病机理尚不完全清楚,但针对其炎性反应的特点,目前临床上多发硬化的治疗多以抗炎和免疫调节为主。IFN-β作为临床上第一个批准的免疫调节药物,对于MS的治疗发挥了重要作用。但是,迄今为止,内源性的IFN-β的来源以及其具体的作用靶细胞仍不是十分清楚。中枢神经系统的炎症微环境、炎症调节以及中枢神经系统细胞对炎症反应的响应程度对于MS/EAE的疾病进展具有重要作用。因此,探索中枢神经系统病灶组织在神经免疫和炎症情况下的反应对于阐明MS/EAE的发病机制并发现新的治疗靶点具有重要意义。在前期的研究中,基于mi RNA多靶点性的特点以及其在众多疾病和生理过程中的重要作用,我们聚焦于EAE疾病中病灶部位mi RNA的表达变化,并以此为基础着重从表观遗传学的角度探讨CNS特异性mi RNA在MS/EAE炎性脱髓鞘和影响髓鞘再生中的作用和机制。取得的研究结果如下:(1)利用200μg MOG35-55免疫8-12周雌性C57BL/6小鼠,发病高峰期(免疫后20天),麻醉后PBS充分灌注小鼠,取EAE小鼠和同龄同性别小鼠脊髓腰膨大段提取总RNA,反转录后用SABiosciences公司的小鼠mi RNA PCR array芯片检测EAE小鼠脊髓病灶部位mi RNA的表达差异,发现EAE小鼠病灶部位mi R-146a、mi R-147、mi R-155、mi R-223、mi R-509-3p的表达显著上调,mi R-124,mi R-132,mi R-338的表达显著下调。选取表达显著上调mi R-146a分子为研究对象,利用RNA原位杂交技术对其表达进行验证,发现EAE小鼠病灶部位的mi R-146a相较于对照组显著升高。进一步分离纯化并体外培养小鼠的原代星形胶质细胞和小胶质细胞,利用IL-17,TNF-α以及IL-17结合TNF-α等炎症因子处理,发现炎症刺激会显著升高两种细胞内mi R-146a的表达,并且IL-17和TNF-α具有协同作用效果。(2)构建带有mi R-146a sponge的用以抑制mi R-146a的表达慢病毒载体,将其命名为mi R-146a inhibitor,包装慢病毒并进行滴度测定。用PBS将病毒稀释成5×108IU/ml。利用200μg MOG35-55免疫8-12周雌性C57BL/6小鼠,发病初期(免疫后第14d)和高峰期(免疫后第20d)利用侧脑室注射的方式分别给予20μl/mouse慢病毒LV-mi R-146a inhibitor或对照慢病毒,统计小鼠临床神经功能评分,发现EAE中中枢抑制mi R-146a的表达可以显著缓解病情。而抑制中枢mi R-146a的表达对外周免疫细胞的比例、数量、增殖、炎症因子的分泌并无影响。免疫组化结果显示抑制中枢mi R-146a的表达减少了中枢炎性浸润和脱髓鞘的变化。同时免疫荧光化学结果显示,抑制中枢mi R-146a的表达显著减少了中枢神经系统白细胞、单核吞噬细胞和中性粒细胞的数量,也缓解了病灶部位的星形胶质细胞化。(3)检测EAE发病不同阶段中枢神经系统病灶部位mi R-146a及相关基因的表达,发现mi R-146a在病灶部位的表达与TRAF6及IFN-β的表达显著负相关。进一步利用生物信息学分析发现,TRAF6基因3’非编码区含有两个mi R-146a结合位点(447-468,506-532),体外培养的星形胶质细胞中,转染mi R-146a会显著抑制TRAF6的表达。利用携带TRAF6 3’非编码区或mi R-146a结合位点突变的TRAF6 3’非编码区双荧光素酶报告基因系统转染原代星形胶质细胞发现TRAF6是mi R-146a的直接作用靶点。进一步利用EAE动物模型发现,中枢特异性抑制mi R-146a表达的同时下调TRAF6的表达阻断了抑制mi R-146a对EAE的保护作用,而下调mi R-146a或过表达TRAF6均能缓解EAE的病情,说明在EAE的CNS病灶区域,mi R-146a至少部分的通过作用于TRAF6调控中枢内源性1型干扰素系统的表达或分泌参与EAE疾病进程。上述结果表明mi R-146a通过调控TRAF6及1型干扰素的表达及分泌参与EAE疾病进程,抑制中枢mi R-146a的表达会显著减轻EAE的病情,为阐明MS/EAE发病机制以及疾病治疗新靶点的发现奠定基础。
[Abstract]:Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS). It is an autoimmune demyelinating disease model of the central nervous system mediated by Th1 and Th17 cells. In the central nervous system, the self activated Th1/Th17 (CD4 +T) cells, CD8+T cells, macrophage / dendritic cells, hypertrophy in the central nervous system. Cells and activated microglia can produce a large number of proinflammatory cytokines IFN- gamma, IL-17, TNF- alpha, GM-CSF and IL-23 to promote cell mediated immune response. Although the pathogenesis is not completely clear, in view of the characteristics of its inflammatory reaction, most of the treatment of multiple sclerosis is mainly based on anti-inflammatory and immunomodulatory treatment of.IFN- beta. The first approved immunomodulatory drugs in the clinic have played an important role in the treatment of MS. However, to date, the source of endogenous IFN- beta and its specific target cells are still not very clear. The inflammatory microenvironment of the central nervous system, the regulation of inflammation, and the response of the central nervous system cells to the inflammatory response It is important for the progress of the MS/EAE disease. Therefore, it is important to explore the response of the central nervous system in the condition of neuroimmunology and inflammation to elucidate the pathogenesis of MS/EAE and to discover new therapeutic targets. In the previous study, the characteristics of multiple targets based on MI RNA and its multitudinous diseases and physiology The important role of the process is to focus on the expression of MI RNA in the lesion site in EAE disease. On this basis, we focus on the role and mechanism of CNS specific mi RNA in MS/EAE inflammatory demyelinating and myelin regeneration from the epigenetic point of view. The results obtained are as follows: (1) the use of 200 mu g MOG35-55 to immunization for 8-12 weeks female C57BL/6 mice were at the peak of onset (20 days after immunization). After anesthesia, PBS was fully perfused in mice. The total RNA was extracted from the spinal cord of the spinal cord of the EAE mice and the same sex mice. The MI RNA PCR array chip of the SABiosciences company was used to detect the difference in the MI RNA in the spinal cord of the spinal cord of the EAE mice. The expression of a, MI R-147, MI R-155, MI R-223, MI R-509-3p significantly up-regulated, MI R-124. The primary astrocytes and microglia were purified and cultured in vitro, and treated with IL-17, TNF- alpha and IL-17 combined with TNF- alpha, the expression of MI R-146a in two cells was significantly increased by inflammatory stimulation, and IL-17 and TNF- alpha had synergistic effects. (2) the construction of MI R-146a sponge was used to inhibit mi. R-146a expressed the lentivirus vector, named it mi R-146a inhibitor, packed the lentivirus and tested the titer. The virus was diluted into 5 x 108IU/ml. with 200 mu g MOG35-55 for 8-12 weeks female C57BL/6 mice, and 20 mu l was given by the lateral ventricle in the early stage (after immunization, 14d) and peak period (after immunization). /mouse lentivirus LV-mi R-146a inhibitor or control lentivirus was used to count the clinical neurological function score of mice. It was found that the expression of central inhibition of MI R-146a in EAE could significantly alleviate the disease. The inhibition of central mi R-146a expression did not affect the proportion of peripheral immune cells, quantity, proliferation, and secretion of inflammatory factors. The expression of MI R-146a in the central nervous system reduced the changes in the central inflammatory infiltration and demyelination. At the same time, the immunofluorescence chemical results showed that the inhibition of the expression of MI R-146a in the central nervous system significantly reduced the number of leukocytes in the central nervous system, the number of monocyte phagocytes and neutrophils, and also the astrocytomization of the site of the disease. (3) detection of the pathogenesis of EAE. The expression of MI R-146a and related genes in the central nervous system of different stages showed that the expression of MI R-146a was negatively correlated with the expression of TRAF6 and IFN- beta. Further use of bioinformatics analysis found that the TRAF6 gene 3 'non coding region contains two mi R-146a junction sites (447-468506-532), and the in vitro culture is star shaped. In glial cells, transfection of MI R-146a significantly inhibits the expression of TRAF6. It is found that TRAF6 is the direct target of MI R-146a using the TRAF6 3 'non coding region double luciferase reporter gene system carrying TRAF6 3' non coding region or MI R-146a binding site mutation, and further using EAE animal model to find the target. The inhibition of the expression of MI R-146a and the expression of TRAF6 inhibited the protective effect of MI R-146a on EAE, and the downregulation of MI R-146a or overexpression of TRAF6 could mitigate the condition of EAE. The results show that MI R-146a is involved in the process of EAE disease through the regulation of the expression and secretion of TRAF6 and type 1 interferon, and the inhibition of the expression of central mi R-146a can significantly reduce the condition of EAE, laying a foundation for the discovery of the pathogenesis of MS/EAE and the discovery of new targets for the treatment of disease.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R744.5

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