中枢神经系统细胞特异性IFN-γ信号通路在实验性自身免疫性脑脊髓炎中的作用机制研究

发布时间：2018-04-08 14:17

本文选题：实验性自身免疫性脑脊髓炎　切入点：多发性硬化症　出处：《陕西师范大学》2015年博士论文

【摘要】：多发性硬化症(Multiple sclerosis, MS)是一种由1型辅助T细胞(Thl)和17型辅助T细胞(Thl7)介导的中枢神经系统自身免疫性炎性脱髓鞘疾病。该病主要累及青壮年,是造成青壮年神经系统功能性残障的最主要因素之一。实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis, EAE)是为了研究多发性硬化症(Multiple sclerosis, MS)而建立的动物模型,对该动物模型的研究表明,Thl细胞的特征性细胞因子IFN-y通过作用于免疫细胞在EAE的疾病发生发展中起着重要的作用,但是其如何作用于中枢神经系统(central nervous system; CNS)的细胞,尤其是在体内情况下其作用效果研究的较少。因此,本研究利用髓鞘少突胶质糖蛋白(Myelin oligodendrocyte glycoprotein, MOG)免疫诱导的EAE动物模型,通过侧脑室注射特异性敲低星形胶质细胞(Astrocyte)或小胶质细胞(microglia)IFN-γ受体(IFN-γR1)表达的方法阻断不同CNS细胞IFN-y信号通路,研究CNS不同细胞特异性IFN-y信号通路在EAE中的作用及其作用机制,以期为更好的理解MS/EAE的病程机制并寻找新的细胞特异性靶点奠定基础。所取得的主要研究结果如下：1.利用免疫荧光双染技术发现,在小鼠脊髓切片中,IFN-γR1在星形胶质细胞、小胶质细胞以及少突胶质细胞中均有表达,神经元中不表达IFN-yr1;2.利用免疫荧光染色及实时荧光定量PCR技术发现在MOG免疫诱导的EAE模型的脊髓和脑组织中,相较于健康小鼠,IFN-γR1呈诱导表达,提示CNS的IFN-γ信号通路可能参与了EAE的病程发生;3.构建基于miR-30的星型胶质细胞或小胶质细胞／单核巨噬细胞特异性IFN-γR1敲低的慢病毒载体,体内体外实验验证,该慢病毒可以有效的敲低IFN-γR1的表达；4.EAE临床神经功能评分提示星形胶质细胞或小胶质细胞特异性沉默IFN-γ信号通路在EAE诱导发病过程中具有截然相反的作用,星形胶质细胞特异性阻断IFN-γ信号通路会减轻疾病的严重程度,然而,小胶质细胞特异性阻断该信号通路则会加重疾病病情；5.通过HE染色,脊髓单个核细胞计数以及流式细胞分析发现,星型胶质细胞特异性阻断IFN-γ信号通路减少了中枢神经系统的炎性细胞浸润,从而使得脱髓鞘程度减轻；6.体内实验发现,阻断星形胶质细胞降低了脊髓中致炎性细胞因子IL-6和IL-17A的表达,同时,趋化因子CXCL2、CXCL12、CCL20和MMP9等的表达显著降低。体外星形胶质细胞原代培养实验发现,敲低IFN-γR1之后,星型胶质细胞在IL-17以及IL-17和TNF刺激下,相应IL-17信号通路的趋化因子CXCL1和CXCL2以及CCL20的表达显著降低,而在IFN-γ或IFN-γ和TNF的刺激下,相应IFN-γ信号通路的化学趋化因子CXCL9、CXCL10以及CXCL11的表达显著下降。化学趋化实验证实,IFN-γR1缺失的星形胶质细胞招募或趋化单个核细胞的能力显著下降；7.阻断小胶质细胞的IFN-γ信号通路促进了中枢的炎性浸润和小胶质细胞的增殖。HE染色、脊髓单个核细胞计数以及流式细胞分析发现小胶质细胞特异性阻断IFN-γ信号通路增加了脑干和脊髓的炎性细胞浸润。实时荧光定量PCR显示,小胶质细胞特异性阻断IFN-γ信号通路对CNS趋化因子表达的影响不明显,IFN-γ的表达量显著降低,而抗炎性细胞因子IL-10的表达量显著降低。免疫荧光染色以及流式细胞分析显示小胶质细胞特异性阻断IFN-γ信号显著增加了CNS小胶质细胞／单核巨噬细胞的数量。8.利用体外培养的原代小胶质细胞发现,IFN-γ处理显著降低了小胶质细胞的数量,而IFN-γ处理对IFN-γR1缺失的小胶质细胞的数量则没有影响。实时荧光定量PCR实验发现,IFN-γ能够促进小胶质细胞趋化因子以及致炎性细胞因子的表达,但其更显著的作用表现在小胶质细胞IFN-γ信号通路能显著控制或抑制该类细胞的数量。进一步分析发现相较于星形胶质细胞,IFN-γ或IFN-γ结合TNF的处理可以显著提高p21的表达并抑制cmyc的表达,提示小胶质细胞上的IFN-γ信号通路通过影响细胞周期来控制细胞的数量。同时caspase免疫荧光染色以及LDH活性测定表明IFN-γ信号通路影响小胶质细胞的数量不是通过促进细胞凋亡或坏死来实现的；9.在发病初期或发病高峰期特异性阻断星形胶质细胞IFN-γ信号通路可以有效缓解Thl细胞和Th17细胞介导的EAE疾病的严重程度。上述结果表明中枢神经系统不同细胞同一信号通路可能在疾病中起着不同的病理作用。星形胶质细胞的IFN-γ信号通路通过影响趋化因子的表达来影响疾病发展,而小胶质细胞的该信号通路则通过影响控制小胶质细胞的数量来影响疾病进程。该研究结果对于进一步理解MS/EAE的病程机制具有重要意义,也有利于发现新的细胞特异性的治疗靶点。
[Abstract]:Multiple sclerosis (Multiple sclerosis MS) is a T cell by helper type 1 (Thl) and type 17 helper T cells (Thl7) mediated by the central nervous system autoimmune inflammatory demyelinating disease. The disease mainly affects young adults, is one of the main factors causing the young nervous system function of disability. The experimental autoimmune encephalomyelitis (Experimental autoimmune, encephalomyelitis, EAE) in order to study multiple sclerosis (Multiple sclerosis, MS) and the establishment of the animal model of the animal model, characteristic cell factor IFN-y Thl cells by acting on immune cells in EAE the disease plays an important role in the development, but its role in the central nervous system (central nervous system; CNS) cells, especially in the case of the in vivo effects of less. Therefore, this study uses the myelin sheath Oligodendrocyte glycoprotein (Myelin oligodendrocyte glycoprotein, MOG EAE) animal model of immune induced, knockdown of astrocytes by intracerebroventricular injection of specific (Astrocyte) or microglia (microglia) IFN- receptor gamma (IFN- gamma R1) expression of CNS cells with methods of blocking IFN-y signal pathway of different cell specific CNS IFN-y signal pathway in the role of EAE and its mechanism, in order to better understand the disease mechanism of MS/EAE and lay the foundation for cell specific new target. The main results are as follows: 1. using immunofluorescence double staining technique found in mouse spinal cord slices, IFN- gamma R1 in astrocytes cells, microglia and oligodendrocytes were expressed, the expression of IFN-yr1 in neurons; 2. by immunofluorescence staining and real-time fluorescence quantitative PCR technology found in EAE model induced by vaccination with MOG The type of spinal cord and brain tissue, compared with healthy mice, IFN- showed R1 expression induced by gamma gamma, suggesting that IFN- signaling pathway of CNS may be involved in the course of EAE; 3. building miR-30 astrocytes or microglia / macrophage specific IFN- gamma R1 lentiviral vector based on knock is low. In vivo and in vitro experiments, the expression of lentivirus can effectively knockdown of IFN- gamma R1; 4.EAE clinical neurological function score showed opposite effects on astrocytes or microglia specific silencing IFN- signal pathway in EAE induced pathogenesis, astrocyte specific blocking severity, IFN- gamma signal pathway will reduce the severity of the disease. However, microglia specific blocking this pathway will aggravate the disease; 5. by HE staining of spinal cord mononuclear cell counting and flow cytometry analysis showed that astrocytes fine Cell specific blocking IFN- signal pathway of gamma reduces CNS inflammatory cell infiltration, so as to reduce the degree of demyelination; 6. in vivo, blocking astrocytes reduced expression of inflammatory cytokines induced by IL-17A and IL-6 in the spinal cord at the same time, chemokine CXCL2, CXCL12, CCL20 and MMP9 expression etc. decreased significantly. Astrocytes cultured in vitro experiments showed that after knockdown of IFN- gamma R1, astrocytes in IL-17 and IL-17 and TNF under the stimulation of the corresponding signal pathway of IL-17 chemokine CXCL1 and CXCL2 and CCL20 expression decreased significantly, while IFN- or IFN- gamma gamma and TNF stimulation the corresponding chemical IFN- signal pathway of gamma chemokine CXCL9, CXCL10 and CXCL11 expression decreased significantly. Chemotactic experiments confirmed that IFN- gamma R1 deficient astrocytes recruitment or chemotaxis of mononuclear cells significantly decreased the ability of; 7. blocking IFN- signal pathway of gamma microglia promote inflammatory CNS infiltration and proliferation of microglial cells with.HE staining, spinal cord mononuclear cell counting and flow cytometry analysis showed that microglia specific blocking IFN- signal pathway of gamma increases the brainstem and spinal cord infiltration of inflammatory cells. Real time fluorescent quantitative PCR display microglia, the blocking effect of IFN- signal pathway on CNS expression of chemokines is not obvious, the expression of IFN- gamma was significantly decreased, whereas the expression of anti-inflammatory cytokines significantly decreased IL-10. Immunofluorescence staining and flow cytometry analysis showed that microglia specific blocking primary microglia IFN- the gamma signal significantly increased the number of.8. CNS microglia / macrophage in vitro showed that IFN- gamma treatment significantly reduced the number of microglia, and IFN- gamma gamma R1 treatment on IFN- It did not affect the number of microglia was found missing. Real time fluorescent quantitative PCR experiments, IFN- gamma can promote the expression of microglia chemokines and proinflammatory cytokines, but the more significant role can significantly control or suppression of these cells in microglia IFN- gamma pathway. Further the analysis found that compared to astrocytes, can significantly increase the expression of p21 and inhibiting the expression of cmyc IFN- or IFN- TNF combined with gamma gamma treatment, suggesting that IFN- number of gamma signal pathway of microglial cells by influencing the cell cycle to control cells. At the same time, caspase staining and LDH activity assay showed that the number of microglia IFN- gamma signaling influences by promoting apoptosis or necrosis is not achieved; 9. in early onset or the peak incidence of specific blocking astrocyte IFN- gamma signal The severity of the road can effectively alleviate the Thl cell and Th17 cell mediated EAE disease. The above results indicate that the central nervous system of different cells in the same signaling pathway may play a role in the pathology of different diseases. The expression of IFN- gamma signaling pathway in astrocytes by influencing the chemotactic factor to influence the development of the disease, and the number of the signal pathway of microglia is affected by control of microglia to influence the disease process. The research result has important significance to further understand the mechanism of the course of MS/EAE, but also conducive to find new therapeutic target cell specificity.

【学位授予单位】：陕西师范大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R744.51

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