黄芪多糖抑制小鼠EAE 及调控BV-2 神经小胶质细胞活化的实验研究

目的:研究黄芪多糖(APS)对实验性自身免疫性脑脊髓膜炎(EAE)的治疗作用以及对参与EAE 发病机制的神经小胶质细胞活化的调控作用及其可能的作用机制。方法:动物实验:MOG35-55 诱导C57BL/6 小鼠建立EAE 模型,予APS 给药干预,通过5 级临床症状评分观察APS 对小鼠EAE 的治疗作用。细胞实验:MTT 法检测脂多糖(LPS)对于BV-2 神经小胶质细胞的增殖抑制作用,筛选合适的LPS 刺激浓度活化神经小胶质细胞,构建BV-2 神经小胶质细胞活化的模型;倒置显微镜观察BV-2 神经小胶质细胞形态学的改变;ELISA 法检测BV-2 神经小胶质细胞IFN-酌、TNF-α的分泌水平变化;观察不同浓度的APS 对BV-2 神经小胶质细胞活化的调控作用;APS 干预后,Western blot、Real-time PCR 方法分别检测BV-2 神经小胶质细胞PD-L1 蛋白和mRNA 表达水平的变化。结果:APS 能够有效治疗小鼠EAE 的临床症状,成功建立了体外BV-2 神经小胶质细胞活化模型,一定浓度的APS 能够抑制BV-2 神经小胶质细胞的活化,提高活化的BV-2 细胞的生存活性,降低IFN-酌、TNF-α的分泌水平,促进活化的BV-2 神经小胶质细胞PD-L1 基因及蛋白表达上调。结论:APS 对小鼠EAE 具有明显的治疗作用,其发挥作用的机制可能是APS 能够有效抑制神经小胶质细胞的活化,降低炎性细胞因子IFN-酌、TNF-α的分泌,对神经小胶质细胞有抗炎保护作用,PD-1/ PD-L1 通路可能是APS 发挥抗炎作用的重要途径。     

从动物模型的视角研究多发性硬化髓鞘保护和再生的小胶质细胞靶点

小胶质细胞是定植于中枢神经系统(CNS)的免疫细胞,构成了CNS的第一道防线。多发性硬化(MS)是以炎症脱髓鞘伴轴突损伤为主要特征的CNS炎症变性疾病,小胶质细胞激活在其发生发展过程中担负着重要角色。在MS动物模型的CNS可见大量激活的小胶质细胞,其功能复杂,主要有促炎症M1表型和抗炎症M2表型两种小胶质细胞,具有破坏和保护髓鞘的双重作用:一方面M1表型小胶质细胞可通过释放促炎因子、自由基等对少突胶质细胞(OLs)及其前体细胞产生损伤,造成髓鞘破坏;另一方面M2表型小胶质细胞还可通过吞噬髓鞘碎片、分泌抗炎及再生因子等作用,加速髓鞘的修复和再生。本文对激活状态下M1/M2小胶质细胞的功能和靶向小胶质细胞转化在经典MS动物模型中的研究进展作一综述,为靶向小胶质细胞治疗CNS脱髓鞘疾病提供基础研究和临床应用的实验依据。     

小胶质细胞的活化在实验性自身免疫性脑脊髓炎中的作用

多发性硬化( multiple sclerosis,MS)是一种常见的以中枢神经系统(central nervous system,CNS)炎性脱髓鞘病变为特征的自身免疫性疾病.MS的病因和发病机制非常复杂,由于病理取材的困难性,在体外建立了该病的动物模型-实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE).人MS及其动物模型EAE发病过程中大量炎性细胞入侵CNS并产生多种致炎因子,脑内微环境受到影响.诸多研究表明在EAE或MS发病中静息态小胶质细胞在浸润的T细胞及释放的细胞因子刺激下发生活化,但它们在EAE中的作用目前尚无统一意见.兹将小胶质细胞在EAE中的活化机制及其在炎症/免疫反应中的作用作一综述.     

嗅鞘细胞移植实验性自身免疫性脑脊髓炎后髓鞘碱性蛋白的表达和小胶质细胞的捕获

目的:研究嗅鞘细胞(OECs)移植实验性自身免疫性脑脊髓炎(EAE)后髓鞘碱性蛋白(Myelin basic protein,MBP)的表达及小胶质细胞捕获OECs情况。方法:用新生近交系Wistar大鼠嗅球培养出OECs,经荧光染料羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)标记后注入同系EAE Wistar大鼠侧脑室,免疫组化检测OECs髓鞘碱性蛋白(MBP)、单核巨噬细胞诱导分子1(ED1)和CFSE共表达情况。结果:培养OECs不表达MBP,经侧脑室移植后在EAE大鼠脑内出现多量MBP+CFSE+细胞,同时在大脑广泛区域及血管周围间隙出现多量ED1+CFSE+细胞,与对照组相比差异显著。结论:OECs在EAE环境下表达MBP分子,其抗原能被脑内小胶质细胞或巨噬细胞捕获。     

重组人髓鞘少突胶质细胞糖蛋白的表达、纯化及免疫原性

目的: 建立人少突胶质细胞糖蛋白细胞外Ig样结构域(MOG^Igd)的硫氧还蛋白(TrxA)融合表达系统,探讨表达产物MOG^Igd-TrxA融合蛋白的免疫原性。方法: (1)应用RT-PCR方法,以人脑组织的总RNA为模板扩增出编码MOG^Igd的cDNA序列,将MOG^Igd cDNA克隆到TrxA融合表达载体pET32a(+)中,重组质粒pET32a-MOG^Igd经表型筛选、酶切鉴定及测序鉴定后,转化至BL21(DEB)trxB^-宿主菌中经异丙硫代钠-β-D-半乳糖苷(IPTG)诱导表达,并通过金属鳌合亲和层析柱进行纯化。(2)C57BL/6小鼠分为MOG^Igd-TrxA组(MOG组)、硫氧还蛋白组(TrxA组)及正常对照组(NC组),12只／组,各组以相应抗原乳剂免疫小鼠制作实验性自身免疫性脑脊髓炎(EAE)模型后观察其临床神经功能和组织病理学改变(HE染色和髓鞘Luxol fast blue染色)评价模型质量。结果: (1)成功获取高产量32 kD MOG^Igd-TrxA融合蛋白,以包涵体形式表达,纯化后纯度达95％左右。(2)MOG组小鼠75％(9/12)发病,于免疫后第(12.14±2.04)d发病,呈慢性非缓解型病程;发病动物组织切片HE染色和髓鞘染色显示不同程度炎性细胞浸润和髓鞘脱失。结论: 成功建立人MOG^Igd的TrxA融合表达系统,表达产物MOG^Igd-TrxA融合蛋白可作为免疫原诱导EAE动物模型。     

CD4~+ T细胞和小胶质细胞在实验性自身免疫性脑脊髓炎中的作用

实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是在动物体内诱导的神经系统慢性脱髓鞘性疾病。EAE的临床表现和病理改变与多发性硬化症(multiple sclerosis,MS)极为相似,是研究MS理想的动物模型。近年来已有研究发现EAE主要是由自身反应性CD4+T细胞介导的自身免疫性脱髓鞘疾病,在EAE动物发病的不同阶段CD4+T细胞和小胶质细胞数量、形态和表型的改变对疾病的进展起重要作用。通过对EAE病理中CD4+T细胞和小胶质细胞的研究,将有助于进一步了解MS的免疫病理特征及发病机制,为MS的治疗和预防奠定基础。     

Fasudil对实验性自身免疫性脑脊髓炎小鼠小胶质细胞和星形胶质细胞的作用

目的:确认法舒地尔(Fasudil)对实验性自身免疫性脑脊髓炎(EAE)的治疗效果,观察法舒地尔对小胶质细胞和星形胶质细胞的作用.方法:成年雌性C57BL/6小鼠用MOG35-55肽免疫制作慢性EAE模型,分别随机在免疫后第3天(Fasudil早期治疗组)和发病时给予Fasudil(Fasudil晚期治疗组),以同样方式给予生理盐水作为对照.观察临床症状和体质量变化;采用免疫荧光组织化学染色、Western blot 法检测脊髓小胶质细胞和星形胶质细胞iNOS和p-NF-κB/p65的表达,ELISA测定脊髓匀浆中IL-1β和TNF-α水平.结果:Fasudil推迟EAE起病,减轻EAE症状,抑制脊髓中小胶质细胞iNOS以及星形胶质细胞p-NF-κB/p65表达,并伴随炎性因子IL-1β和TNF-α释放降低.结论:Fasudil可抑制EAE小鼠小胶质细胞和星形胶质细胞炎性分子的释放.     

斑马鱼——髓鞘损伤与再生研究的理想动物模型

脱髓鞘疾病是以多发性硬化为主要表现的自身免疫性疾病,髓鞘脱失与再生的机制不明,利用动物模型进行深入研究是一个有效途径.新型模式动物——斑马鱼具有发育快速、胚胎透明、遗传学技术成熟等优势,通过构建转基因斑马鱼可以对细胞进行活体、动态观察和基因表达模式分析,是研究髓鞘损伤与再生的理想动物模型.     

小胶质细胞在脑缺血中的作用

19世纪末，Nissal首次描述了小胶质细胞。1919年，西班牙学Del Rino-Htortega用碳酸银染法将小胶质细胞与神经细胞及其它胶质细胞加以区分，推测小胶质细胞在胚胎早期源于中胚层的骨髓前期细胞，即单核吞噬系统。成年期，这些细胞位于中枢神经系统实质，成为小胶质细胞。这些观点现已普遍被人们所接受。根据细胞的形态，小胶质细胞有两种明显不同的亚型：分枝小胶质细胞和阿米巴样小胶质细胞。分枝小胶质细胞，其形态特征为胞体小，具有伸向各个方向的突起，在正常情况下，小胶质细胞为该型，     

Severity of symptoms and demyelination in MOG-induced EAE depends on TNFR1

The individual role of tumor necrosis factor receptor 1 (TNFR1) and TNFR2 signaling in experimental autoimmune encephalomeylitis (EAE) was investigated using mice lacking TNFR1 (TNFR1-/-), TNFR2 (TNFR2-/-) as well as double receptor (TNFR1/2-/-) and double ligand (TNF/LT alpha-/-) knockout mice. In wild-type (wt) mice immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 the clinical course is characterized by an acute disease onset with peak disease scores and a consecutive chronic phase lasting up to 60 days. Compared to control mice, TNF/LT alpha-deficient mice showed a significant delay in disease onset and a remarkable reduction in demyelination which was, however, associated with increased inflammation. In TNFR1-/- and TNFR1/2-/- mice, the disease course was comparable to TNF/LT alpha-deficient mice but rather monophasic and less severe at late time points. Likewise only minimal spinal cord demyelination became apparent. In contrast, the course of EAE in TNFR2-/- mice was severe and associated with remarkable demyelination. Taken together these findings define TNFR1 as crucial mediator in MOG-induced EAE and suggest a protective role for TNFR2 signaling in the clinical course of EAE.     

Olig对少突胶质细胞介导脱髓鞘大鼠髓鞘再生的影响

目的 探讨Olig在少突胶质细胞介导的脱髓鞘大鼠髓鞘再生中的作用。方法 40只健康Wistar大鼠,随机分成正常组、模型对照组、模型组、实验组,用形态学观察和免疫组织化学检测Olig1和Olig2的表达。结果 正常组Olig1位于少突胶质细胞的胞质,模型组胞核表达明显增多,实验组Oligl又重新转移至胞质;正常组Olig2表达位于胞核,模型组中有少量表达于胞质。 结论 在Olig1和Olig2同时缺失时,导致全脑不能形成少突胶质细胞,严重影响髓鞘的再生。     

The neurotoxicant, cuprizone, as a model to study demyelination and remyelination in the central nervous system

Myelin of the adult CNS is vulnerable to a variety of metabolic, toxic, and autoimmune insults. That remyelination can ensue, following demyelinating insult, has been well demonstrated. Details of the process of remyelination are, however difficult to ascertain since in most experimental models of demyelination/remyelination the severity, localization of lesion site, or time course of the pathophysiology is variable from animal to animal. In contrast, an experimental model in which massive demyelination can be reproducibly induced in large areas of mouse brain is exposure to the copper chelator, cuprizone, in the diet. We review work from several laboratories over the past 3 decades, with emphasis on our own recent studies, which suggest an overall picture of cellular events involved in demyelination/remyelination. When 8 week old C57BL/6 mice are fed 0.2% cuprizone in the diet, mature olidgodendroglia are specifically insulted (cannot fulfill the metabolic demand of support of vast amounts of myelin) and go through apoptosis. This is closely followed by recruitment of microglia and phagoctytosis of myelin. Studies of myelin gene expression, coordinated with morphological studies, indicate that even in the face of continued metabolic challenge, oligodendroglial progenitor cells proliferate and invade demyelinated areas. If the cuprizone challenge is terminated, an almost complete remyelination takes place in a matter of weeks. Communication between different cell types by soluble factors may be inferred. This material is presented in the context of a model compatible with present data -- and which can be tested more rigorously with the cuprizone model. The reproducibility of the model indicates that it may allow for testing of manipulations (e.g. available knockouts or transgenics on the common genetic background, or pharmacological treatments) which may accelerate or repress the process of demyelination and or remyelination.     

Implications of protease M/neurosin in myelination during experimental demyelination and remyelination

Protease M/neurosin is a serine protease expressed by oligodendrocytes (OLGs) in the central nervous system (CNS). To investigate the role of protease M/neurosin during experimental demyelination and remyelination, mice were fed cuprizone (bis-cyclohexanon oxaldihydrazone). Semi-quantitative RT-PCR analysis and immunohistochemistry revealed that the expressions of protease M/neurosin mRNA and protein were rapidly reduced in demyelination, whereas the expression of protease M/neurosin was increased in pi form of glutathione-S-transferases (GST-pi)-positive OLGs during remyelination. Cultured primary OLGs displayed a strong correlation between protease M/neurosin and myelin basic protein (MBP). After tumor necrosis factor-alpha (TNF-alpha) and IFN-gamma stimulation, these proteins showed colocalization in the oligodendroglial process. The suppression of protease M/neurosin using RNAi reduced the level of MBP mRNA in cultured OLGs. In contrast, the reduced level of protease M/neurosin was not associated with oligodendroglial cell death or differentiation in cultured OLGs. This study identifies that protease M/neurosin in OLGs is closely associated with the expression of the MBP and the PLP gene. Our data emphasize that the maintenance of myelination is an important function of protease M/neurosin in OLGs, suggesting its relation to the oligodendroglial response to myelin disorders.     

Effectors of demyelination and remyelination in the CNS: implications for multiple sclerosis

Most of the research on multiple sclerosis (MS) has focused on the early events that trigger demyelination and subsequent remyelination. Less attention has been given to the factors that directly mediate the demyelination that is the hallmark of the disease. Effector cells or molecules are those factors directly responsible for mediating the damage in the disease. Similarly, there are effector molecules that are critical for remyelination in the central nervous system (CNS). By understanding those effector molecules in demyelination and remyelination that directly influence the pathologic process, we should be able to generate specific therapies with the greatest potential for benefiting MS patients. This review focuses on effector cells and molecules that are critical for demyelination and remyelination in MS but also in experimental models of the disease including experimental autoimmune encephalomyelitis (EAE), virus-induced models of demyelination (Theiler's virus, murine hepatitis virus), and toxic models of demyelination (lysolecithin, ethidium bromide, and cuprizone). These are models in which the effector molecules for demyelination and remyelination have been most precisely evaluated.     

Myelin water imaging to detect demyelination and remyelination and its validation in pathology

Damage to myelin is a key feature of multiple sclerosis (MS) pathology. Magnetic resonance imaging (MRI) has revolutionized our ability to detect and monitor MS pathology in vivo. Proton density, T₁ and T₂ can provide qualitative contrast weightings that yield superb in vivo visualization of central nervous system tissue and have proved invaluable as diagnostic and patient management tools in MS. However, standard clinical MR methods are not specific to the types of tissue damage they visualize, and they cannot detect subtle abnormalities in tissue that appears otherwise normal on conventional MRIs. Myelin water imaging is an MR method that provides in vivo measurement of myelin. Histological validation work in both human brain and spinal cord tissue demonstrates a strong correlation between myelin water and staining for myelin, validating myelin water as a marker for myelin. Myelin water varies throughout the brain and spinal cord in healthy controls, and shows good intra‐ and inter‐site reproducibility. MS plaques show variably decreased myelin water fraction, with older lesions demonstrating the greatest myelin loss. Longitudinal study of myelin water can provide insights into the dynamics of demyelination and remyelination in plaques. Normal appearing brain and spinal cord tissues show reduced myelin water, an abnormality which becomes progressively more evident over a timescale of years. Diffusely abnormal white matter, which is evident in 20%–25% of MS patients, also shows reduced myelin water both in vivo and postmortem, and appears to originate from a primary lipid abnormality with relative preservation of myelin proteins. Active research is ongoing in the quest to refine our ability to image myelin and its perturbations in MS and other disorders of the myelin sheath.     

Return of axonal and glial membrane specializations during remyelination after tellurium-induced demyelination

Summary We have studied remyelination of rat peripheral nerves after tellurium-induced demyelination using thin section and freeze-fracture techniques. In rats fed a 1% tellurium diet, regions of demyelination were readily identified by myelin debris and the presence of large denuded axons. Remyelination occurred despite continued tellurium ingestion. However, the demyelinated axons underwent a more rapid remyelination if tellurium was removed from the diet. Remyelination proceeded as described for myelination in the normal developing animal. Sites destined to become nodes of Ranvier were identified as patches of intramembranous particles in the axonal E-face. Early terminal loops of the remyelinating Schwann cell were found adjacent to these particle patches. As wrapping proceeded, terminal loops of myelin, along with associated rows of dimeric-particles characteristic of the axonal P-face, were wound into a paranodal location. This winding of the membrane specializations and associated terminal loops resulted in the reformation of morphologically normal paranodes. The size of the nodal E-face particle patch increased in concordance with increases in the number of paranodal loops until an annulus of particles was obtained as seen in the normal node. The thin section and freeze-fracture morphology of remyelinated fibres was indistinguishable from the morphology of control fibres. These observations are discussed with respect to proposed functions of membrane specializations in myelination and nerve conduction.     

Complement C5 in experimental autoimmune encephalomyelitis (EAE) facilitates remyelination and prevents gliosis

Activation of the classical complement system is known to play a central role in autoimmune demyelination. We have analyzed the role of complement component C5 in experimental autoimmune encephalomyelitis (EAE) using C5-deficient (C5-d) and C5-sufficient (C5-s) mice. Both groups of mice displayed early onset EAE, a short recovery phase, and similar stable chronic courses. However, in contrast to the clinical similarities, marked differences were apparent by histopathology. During acute EAE in C5-d, a delay in inflammatory cell infiltration and tissue damage was observed along with restricted lesion areas, which in C5-s mice were more extensive and diffuse. More striking were the differences in chronic lesions. In C5-d mice, inflammatory demyelination and Wallerian degeneration were followed by axonal depletion and severe gliosis, while in C5-s, the same initial signs were followed by axonal sparing and extensive remyelination. In C5-d, immunohistochemistry and Western blotting showed an increase in glial fibrillary acidic protein and a decrease in neurofilament protein, proteolipid protein, and several pro-inflammatory markers. These results in the EAE model indicate that absence of C5 resulted in fiber loss and extensive scarring, whereas presence of C5-favored axonal survival and more efficient remyelination.