【作者】 李智；

【导师】 何小华；

【作者基本信息】 武汉大学 ， 病理学与病理生理学， 2013， 博士

【摘要】 效应记忆T细胞(TEM)上的Kv1.3电压门控钾离子通道是一个治疗神经免疫性疾病多发性硬化症(MS)的新靶点。在前期研究中,我们获得了一种新型的靶向作用于Kvl.3通道的多肽类阻断剂,命名为ADWX-1,该药物能高效性高选择性地阻断T细胞上的Kv1.3离子通道。我们将其运用于多发性硬化的动物模型—实验性自身免疫性脑脊髓炎(EAE),检测其对EAE模型大鼠的治疗作用并探讨其作用机制。靶向Kv1.3多肽主要通过抑制CD4+CCR7表型的TEM细胞激活来有效治疗EAE模型大鼠。在体内实验中,多肽能降低模型鼠的行为学评分,通过H.E.染色,LFB染色,透射电镜等组织学检测,多肽能减少中枢神经的炎性浸润与脱髓鞘病变,轴索损伤。多肽的神经保护作用可能与其对CD4+T细胞的免疫调节功能有关,ELISA检测结果表明,多肽能降低模型鼠血清的IL-2, IFN-γ TNF-α等Thl类炎性细胞因字的分泌,多肽亦能降低模型鼠中枢神经组织的IL-2分泌。在体外实验中,通过ELISA检测细胞激活后的IL-2分泌水平,多肽抑制了人Jurkat T细胞和模型鼠PBMC细胞的激活,并进一步抑制了模型鼠PBMC细胞的增殖。通过体外流式分选结合ELISA检测活化后细胞因子分泌的方法,多肽能选择性抑制EAE模型鼠中具有CD4+CCR7表型的TEM细胞的活化增殖,而对CD4+CCR7+细胞作用并不明显,维持了这一类细胞在病理状态下的正常免疫功能,有效避免了过度免疫所产生的副作用。为了阐明多肽调节CD4+CCR7细胞激活的作用机制,首先采用流式分选了这一亚型细胞并给予多肽药物干预,通过荧光分光光度法检测Ca2+信号；然后给予Ca2+信号通路及PKCθ信号通路相关的阻断剂,检测IL-2激活诱导的细胞增殖；进一步采用Western-blotting检测NF-κB的激活信号,研究发现多肽参与了TCR信号转导过程中IL-2通路的激活,多肽除了通过传统的Ca2+信号途径即Kv1.3-Ca2+-NFAT途径改变IL-2信号外,还能通过调节Kv1.3-PKCθ-NF-κB轴线来影响IL-2激活信号。通过RT-PCR及流式细胞术,发现在CD4+CCR7细胞激活过程中伴随了Kv1.3mRNA和蛋白表达的上调,而给予多肽可以减少Kv1.3基因的高表达,多肽对通道表达的调控作用在CD4+CCR7+细胞中并不明显,进一步支持了多肽的选择性作用。由于CD4+CCR7细胞依赖Kv1.3通道活性调节其激活信号,多肽对CD4+CCR7细胞中Kv1.3基因表达的选择性调控影响了细胞的再激活,多肽能够抑制再激活过程中的钙信号与IL-2分泌。多肽对Kv1.3表达及功能的抑制使其对这一表型的细胞激活具有一个长期的抑制效果,有效控制了炎性细胞的大量产生。另外,通过流式检测IL-17+细胞数量和ELISA检测IL-17类细胞因子发现多肽调节了分化后的Th17细胞再激活,但是并不影响细胞因子诱导的Thl7分化过程,这是基于分化后的Thl7细胞具有CCR7-的表型。多肽对CD8+T细胞具有类似作用,多肽可以选择性抑制CD8+CCR7细胞激活后的炎性细胞因子颗粒酶B (GrB)分泌,但是对CD8+CCR7+细胞激活后的GrB分泌并无明显作用,在体内多肽阻止了模型鼠由于CD8+T细胞激活所引起的GrB分泌,进而缓解了细胞毒性的GrB对神经少突胶质细胞损伤,减少了髓鞘蛋白的过量消耗,具有间接的神经保护作用。靶向Kv1.3多肽在EAE模型中具有多种调节功能,主要为选择性抑制Kv1.3高表达的CD4+Effector TEM细胞激活机制,这一效应通过细胞激活过程中多肽作用的两种不同模式来实现：一方面多肽调节了Kv1.3通道的活性及IL-2信号通路,另一方面多肽调控了Kv1.3通道的基因,这些证据提示ADWX-1作为靶向针对T淋巴细胞Kv1.3通道的多肽类阻断剂是一类具有临床应用价值的新型免疫调节剂,对MS等神经免疫性疾病的治疗具有重要意义。更多还原

【Abstract】 The voltage-gated Kvl.3K+channel in effector memory T cells (TEM) serves as a new therapeutic target for multiple sclerosis (MS). In our previous studies, the novel peptide ADWX-1was designed and synthesized as a specific Kvl.3blocker. This peptide has a high potency and high selectivity toward Kv1.3channel. However, it is unclear if and how ADWX-1alleviates experimental autoimmune encephalomyelitis (EAE), a model for MS.In this study, the administration of ADWX-1significantly ameliorated the rat EAE model by selectively inhibiting CD4+CCR7-phenotype TEM cell activation. In vivo the peptide can decrease the clinical score of the EAE rats, and reduce the inflammatory infiltration, demyelination and axonal loss in the central nervous system (CNS) by histological study including the H.E. stain, the LFB stain and the Transmission Electron Microscopy (TEM). This neuroprotective effect of the peptide might be attributed to its immunomodulation on CD4+T cells, Therefore series of inflammatory cytokines were analyzed by ELISA. Results showed that the peptide can suppress the serum Th1type cytokines productions in EAE model including IL-2, IFN-γ, and TNF-α The peptide also reduced IL-2secretion in the CNS of EAE rats. In vitro study showed that the peptide inhibited the activation of Jurkat T cells and EAE PBMCs by measuring the IL-2levels after activation. And the peptide further suppressed the proliferation of EAE PBMCs.Further more we elucidated the underlying mechanism of this afficacy. By the methods of ELISA combined with FCAS to detect the cytokines secretions after antigen activation, the peptide selectively inhibit CD4+CCR7" phenotype TEM cell activation and proliferation in vitro. In contrast, the Kvl.3-specific peptide had little effect on CD4+CCR7+cells, therefore limiting side effects. Furthermore, we determined that ADWX-1is involved in the regulation of NF-kB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2pathway of CD4+CCR7-cells. The elevated expressions of Kvl.3mRNA and protein in activated CD4+CCR7-cells were reduced by ADWX-1engagement; however, an apparent alteration in CD4+CCR7+cells was not observed. Moreover, the selective regulation of the Kv1.3channel gene expression pattern by ADWX-1provided a further and sustained inhibition of the CD4+CCR7-phenotype, which depends on the activity of K.v1.3to modulate its activation signal. In addition, ADWX-1mediated the activation of differentiated Th17cells through the CCR7-phenotype, but having no obvious effect on the differentiation process induced by cytokines. Similarly we found the effect of the peptide on CD8+T cells; it selectively suppressed the GrB secretion from CD8+CCR7-cells rather than the CD8+CCR7+cells, in vivo the peptide prevented the GrB production in EAE, the alleviated the neural injury to oligodendrocytes by GrB, reducing the over-exhaust of myelin proteins.The efficacy of ADWX-1is supported by multiple functions, which are based on a Kv1.3high CD4+CCR7-T cell selectivity through two different pathways including the classic channel activity-associated IL-2pathway and the new Kvl.3channel gene expression pathway. This study makes the peptide a promising candidate drug for MS therapy.更多还原