脑梗塞髓鞘蛋白反应性T淋巴细胞的动态观察

目的　脑梗塞急性期脑脊液和血液中存在髓鞘蛋白反应性 Ｔ 淋巴细胞的增殖，观察疾病过程中的 Ｔ 淋巴细胞的连续变化，可能有助于进一步认识其作用机理。方法　采用 Ｅ Ｌ Ｉ Ｓ Ｐ Ｏ Ｔ 方法检测脑梗塞发病后连续两个阶段的病人外周血特异性髓鞘抗原 Ｍ Ｂ Ｐ 反应性 Ｔ 淋巴细胞所分泌的 Ｉ Ｆ Ｎγ的数量，并设立 β Ｂ Ｂ Ｐ作为对照抗原。结果　在这两个连续阶段， Ｍ Ｂ Ｐ反应性 Ｔ 淋巴细胞酶点数均高于对照抗原，差异显著，而在这两个阶段的 Ｍ Ｂ Ｐ反应性 Ｔ 淋巴细胞酶点数之间差异无显著性。结论　脑梗塞发病后，其外周血中髓鞘蛋白反应性 Ｔ 淋巴细胞增殖持续的时间较长，这种现象可能代表与脑组织损伤或再生有关的免疫防御机理。     

脑梗塞髓鞘蛋白反应T淋巴细胞的动态观察

目的 脑梗塞急性期脑脊液和血液中存在髓鞘蛋白反应性Ｔ淋巴细胞的增殖，观察疾病过程中的Ｔ淋巴细胞的连续变化，可能有助于进一步认识其作用机理。方法 采用ＥＬＩＳＰＯＴ方法脑梗塞发病后连续两个阶段的病人外周血特异性髓鞘抗原ＭＢＰ反应性Ｔ淋巴细胞所分泌的ＩＦＮ－γ的数量，并设立β－ＢＢＰ作为对照抗原。结果 在这两个连续阶段，ＭＢＰ反应性Ｔ淋巴细胞酶点数同于对照抗原，差异显著，而在这两个阶段段的ＭＢＰ反应性     

多发性硬化T淋巴细胞系增殖反应性的研究

目的 比较多发性硬化(MS)患者和正常人T淋巴细胞对碱性髓鞘蛋白(MBP)和蛋白脂质蛋白(PLP)及其合成多肽的增殖反应性。方法 选HLA-DRl5亚型MS患者和正常人外周血单个核细胞。以人神经髓鞘为致敏原，采用二次致敏诱导法。经短期体外培养产生外周血T淋巴细胞系(TCL)，用MBP、PLP及其合成多肽等11种抗原检测TCL的增殖反应。结果 MS和对照组对神经髓鞘成分反应均显示多态性。MS组对7种。对照组对5种抗原有反应；比较两组总的平均阳性孔无差别(P=0．177)；两组间对MBP、P30-49、P95-117、P178-191的增殖反应差异有统计学意义。结论 作为神经髓鞘主要成分的MBP和PLP免疫反应性高，可能在体内触发自身免疫反应过程发挥作用，而M87-106、P40-60、P95-117和P185-206等位点值得进一步研究。     

神经干细胞移植促进鼠脊髓损伤后髓鞘结构的修复

目的 观察神经干细胞移植治疗对鼠脊髓损伤后髓鞘结构修复的作用并探讨其作用机制。方法 制备鼠T10脊髓损伤模型,体外培养、诱导鼠神经干细胞,定量评价神经干细胞移植对脊髓损伤后髓鞘结构修复的影响。结果 与对照组相比,神经干细胞移植组明显地增强了蛋白前脂蛋白信使核糖核酸(PLP mRNA)的表达,促进了髓鞘碱性蛋白(MBP)性的髓鞘再生和髓鞘结构的修复。结论 神经干细胞移植通过增强髓鞘的再生而促进了脊髓损伤后髓鞘结构的修复,是急性脊髓损伤一种有效的治疗方案。     

脑梗塞髓鞘蛋白反应性IFN—γ的检测

通过２４例脑梗塞病人和２１例正常对照外周血淋巴细胞进行体外培养，检测其ＭＢＰ和ＭＰＬＰ反应性Ｔ淋巴细胞所分泌的ＩＦＮ－γ，结果发现实验组ＭＢＰ和ＭＰＬＰ反应性ＩＦＮ－γ的产生均高于对照组，差异显著（Ｐ值分别小于０．００２５和０．０２５），提示脑梗塞发生后缺血所致的脑组织的破坏使髓鞘成分作为隐蔽抗原释放，导致相应的髓鞘蛋白反应性Ｔ淋巴细胞的增殖，这些Ｔ细胞及其所分泌的细胞因子可能是脑缺血后造成脑组织损害的第二位因素。     

载脂蛋白E拟肽对EAE小鼠的髓鞘和轴突损伤的影响

目的探讨载脂蛋白E(ApoE)拟肽对实验性自身免疫性脑脊髓炎(EAE)小鼠中枢神经系统髓鞘脱失和轴突损伤的影响。方法以髓鞘少突胶质细胞糖蛋白多肽(MOG35-55)为抗原建立EAE模型。将40只雌性C57BL/6J小鼠随机分为4组,即正常组、EAE组、正常治疗组和EAE治疗组,两个治疗组皮下注射ApoE拟肽。免疫组化法检测髓鞘碱性蛋白(MBP)和神经丝轻链(NF-L)的表达。结果 EAE治疗组中脑和脊髓的MBP和NF-L的表达均高于EAE组(P<0.05)。结论 ApoE拟肽可能对EAE的髓鞘和轴突的损伤有保护作用。     

脑梗死髓鞘蛋白反应性IFN-γ的动态检测

目的了解脑梗死急性期和急性期后体内髓鞘蛋白反应性T淋巴细胞增殖的情况.方法用免疫酶点方法检测脑梗死发病后连续两个阶段外周血特异性MBP反应性IFN-γ酶点数.结果两个阶段酶点数均高于对照抗原,两个阶段之间差异无显著性.结论脑梗死后其外周血中髓鞘蛋白反应性T淋巴细胞增殖持续时间较长,这种现象可能代表与脑组织损伤或再生有关的免疫防御机制.     

神经髓鞘及脱脂神经髓鞘诱导MS-TCL增殖反应比较

目的 比较神经髓鞘和脱脂神经髓鞘诱导多发性硬化(MS)T淋巴细胞系(TCL)对11种神经髓鞘成份的增殖反应。方法 以2种人神经髓鞘在体外二次致敏,诱导MS-TCL和正常人TCL,用MBP、PLP及其合成多肽等抗原检测PTL的增殖反应。结果 与非脱脂TCL相比,脱脂髓鞘使MS组的免疫反应性显著改变。尤其对PLP6种多肽反应性的改变有统计学差异,总平均阳性孔比较P<0.001(3.41±4.83 vs 5.49±5.31)。结论 髓鞘脱脂使MS组增殖反应显著增加,二组的差别更明显。提示在髓鞘脱脂方面MS和正常人反应的异质性,此点对理解MS的病理机制可能很重要。     

多发性硬化患者周围血单核细胞的IL-10分泌细胞变化及意义

目的通过计数分泌细胞因子白细胞介素１０（ＩＬ１０），检查多发性硬化（ＭＳ）患者活动期和缓解期外周血单核淋巴细胞ＩＬ１０分泌细胞水平。方法将单核细胞暴露于中枢神经系统髓鞘素抗原髓鞘碱性蛋白中进行体外短时间培养，用酶联免疫斑点试验（ＥＬＩＳＰＯＴ）检测ＩＬ１０分泌细胞，同时检测其他神经病（ＯＮＤ）及健康对照组。结果显示ＭＳ患者ＩＬ１０分泌细胞缓解期高于活动期。结论认为ＩＬ１０对ＭＳ可能有保护作用。     

髓鞘形成与损伤相关疾病的研究进展 及其与创伤后应激障碍的关联

少突胶质细胞是形成中枢神经系统髓鞘的细胞,有研究表明,少突胶质细胞的细胞骨架 在髓鞘的形成过程中发挥了重要作用。少突胶质细胞的独特易损性所导致的髓鞘脱失在某些疾病中具 有核心地位。与髓鞘损伤相关的指标有髓鞘碱性蛋白（MBP）、髓鞘相关糖蛋白（MAG）、少突胶质细胞转 录因子（OLIG2）等蛋白。与髓鞘损伤所相关的疾病有视神经脊髓炎、多发性硬化、阿尔茨海默病等一些 神经退行性病变以及抑郁症。创伤后应激障碍（PTSD）作为神经精神类疾病的一种,近年来其发病机制、 治疗及预后受到广大学者的关注,但与之相关的因素仍有待于进一步深入探索与研究。现将从髓鞘形 成、髓鞘损伤相关疾病的机制、各个指标在髓鞘损伤所对应的疾病中的具体变化等方面来看髓鞘的形 成与损伤对 PTSD 的发展、恢复的影响。     

Preparation of a novel monoclonal antibody specific for myelin basic protein phosphorylated on Thr

Phosphorylation is one of a number of post-translational modifications resulting in charge microheterogeneity of myelin basic protein (MBP). This phosphorylation is claimed to destabilise the compact myelin sheath by decreasing the interaction of membrane bilayers, thereby creating or maintaining pockets of cytoplasm. To further investigate and localise MBP phosphorylation to discrete regions of the myelin sheath we raised a monoclonal antibody with specificity for a known phosphorylation site in MBP. A synthetic peptide was made by Fmoc peptide chemistry and phosphorylation of Thr⁹⁸ was achieved on the resin by the global phosphorylation methodology, utilising dibenzyl-N,N-diethylphosphoramidite phosphitylation and t-butylhydroperoxideoxidation. The peptide coupled to tuberculin was used to immunise mice for monoclonal antibody production. The selected hybridoma (Clone P12) secreted an IgG2a antibody which reacted strongly with the phosphorylated immunogen and with phosphorylated fractions of bovine MBP obtained by ion exchange chromatography. The antibody had minimal reactivity with the unphosphorylated peptide; the same peptide phosphorylated at another site Ser¹⁰²; a preparation of unphosphorylated MBP obtained by ion exchange chromatography; and with an irrelevant phosphorylated protein (histone). Similar phosphorylation state-specific monoclonal antibodies could be made to recognise other specific phosphorylation sites in MBP or other proteins. It is planned to use these antibodies to quantify and locate the extent of MBP phosphorylation in normal and multiple sclerosis myelin.     

T and B cell responses to myelin basic protein and encephalitogenic epitopes

The major encephalitogenic epitope of myelin basic protein (MBP) for the Lewis rat includes residues 68–84, although a minor epitope has been localized to MBP residues 87–99. We synthesized MBP68–84 and MBP87–99, and immunized rats with these peptides or with MBP in complete Freund's adjuvant (CFA). MBP and MBP68–84 induced paralytic experimental autoimmune encephalomyelitis (EAE) at equimolar concentrations, whereas significantly higher dosages of MBP87–99 were required to elicit paralytic disease. Spleen cells (SpC) from MBP- or MBP68–84-immunized rats could be activated with either MBP or MBP68–84 to transfer EAE to recipients. Anti-MBP antibodies were detected by ELISA in rats immunized with MBP-CFA, and anti-MBP68–84 specific antibodies were present in serum obtained from MBP68–84-immunized animals. However, these antibodies were non-cross reactive. MBP87–99 elicited only a meager antibody response to the immunizing peptide, and cross reactivity with MBP was not observed. Thus, although MBP and each peptide exhibited encephalitogenic activity, and MBP and MBP68–84 were cross reactive at the T cell level, the absence of cross reactivity at the humoral level indicates that significant immunological differences exist between MBP and the synthetic determinants, which may reflect differences in epitope recognition by T and B lymphocytes.     

Identification of a mitogen-activated protein kinase site in human myelin basic protein in situ

Ultrastructural localization of a specific phosphorylated isomer of myelin basic protein (MBP) has been achieved with a monoclonal antibody specific for human MBP sequence, 89–105, in which Thr⁹⁸ was phosphorylated. Cryosections of human brain white matter revealed that gold particles were found localized almost exclusively to the major dense line demonstrating that threonine 98 in the sequence Thr-Pro-Arg-Thr-Pro-Pro-Pro, a mitogen-activated protein kinase-specific site, was phosphorylated in vivo. In two cases of multiple sclerosis, the density of gold particles in myelin was reduced by about 30%, in one case by 42%, and by 80% in a fourth case. However, gold labelling was seen in areas of demyelination suggesting that the phosphorylated threonyl peptide was protected from degradation.     

The proximal peripheral nervous system is a major site of demyelination in experimental autoimmune encephalomyelitis induced in the Lewis rat by a myelin basic protein-specific T cell clone

Experimental autoimmune encephalomyelitis (EAE) was induced in the Lewis rat by the passive transfer of a cytotoxic CD4⁺ T cell clone specific for the 72–89 peptide of guinea-pig myelin basic protein (MBP). Histological studies on rats with neurological signs showed that inflammation was present in the proximal peripheral nervous system (PNS), namely the spinal roots, as well as in the central nervous system (CNS). The main sites of demyelination were the spinal roots in the PNS, and the spinal cord root entry and exit zones in the CNS. The major involvement of the proximal PNS in autoimmune disease directed at MBP is in marked contrast to EAE induced by immunisation with myelin proteolipid protein, where the inflammation and demyelination are restricted to the CNS. These findings may have implications for the human inflammatory demyelinating diseases including multiple sclerosis, in which MBP is a putative target antigen.     

Ultrastructural identification of oligodendrocyte/myelin proteins in corpus callosum of hypothyroid animals

Thyroid hormone (T3) deficiency impairs the development of the CNS, particularly myelination. We have previously described an increase in the frequency of morphological abnormalities in the central myelin sheath in a hypothyroidism model, which reinforced the hypothesis of a role for T3 in myelin compaction. However, there are no data concerning the cellular distribution of myelin proteins in hypothyroid animals. In the present work, we describe the distribution of 2′,3′-cyclic nucleotide 3′-phosphodiesterase (CNPase), myelin basic protein (MBP) and proteolipid protein (PLP) throughout the central myelin sheath of a hypothyroidism model. We used euthyroid and hypothyroid adult rats at 90 days of age. In order to induce hypothyroid status, animals received 0.02% methimazol from the 19th gestation day onwards. After perfusion with a fixative mixture, small pieces of corpus callosum were obtained, dehydrated and embedded in LR White resin. Ultrathin sections were immunoreacted, using specific antibodies revealed by a secondary antibody coupled to colloidal gold particles of 10 nm. Gold particle density per region of myelin sheath for each one of these proteins was obtained. In normal animals, CNPase, PLP and MBP were identified in sites that had already been described in previous studies. In hypothyroid animals, CNPase was identified in the region corresponding to compact lamellae, which normally does not contain this protein, while, in this same region, PLP and MBP immunolabeling were decreased. These results suggest that thyroid hormone deficiency impairs the distribution of the major oligodendrocyte/myelin markers. This effect may justify the reduction in myelin sheath compaction previously demonstrated in a similar model of hypothyroidism.     

冰片对中枢及周围神经髓鞘的影响

目的 探讨冰片对大鼠中枢及周围神经髓鞘的影响.方法 SD大鼠根据灌服冰片石蜡油溶液的剂量不同分为低剂量(10 mL/kg)及高剂量(13mL/kg)2组,每组18只.另设对照组18只,灌服10 mL/kg石蜡油溶液.3组大鼠灌服冰片或石蜡油溶液后24 h、48 h及72 h取血并处死取脑、坐骨神经.进行血清髓鞘碱性蛋白(MAP)检测及透射电镜观察.结果 3个观测时间点中,高、低剂量组大鼠MBP含量明显升高,与对照组比较差异有统计学意义(P<0.05),但高、低剂量组之间差异无统计学意义(P>0.05).灌服冰片后24 h MBP含量最高,随时间延长递减,各时间点之间比较差异有统计学意义(P<0.05).电子显微镜观察大鼠灌服冰片后各时间点均可见中枢神经髓鞘异常,出现外形不规则,板层之间裂开,宽窄不均,纤维缠结现象,以24h改变最明显,随时间延长异常减轻;坐骨神经髓鞘仅在灌服冰片后24 h髓鞘数量出现明显减少,其余未见异常.结论 冰片可引起大鼠中枢及周围神经髓鞘出现一定程度的异常.

Abstract：

Objective To study the effect of borneol on myelin of the central and peripheral nervous systems. Methods The rats were divided randomly into low-dose borneol treatment group (10 mL/kg,n=18) and high-dose borneol treatment group (13 mL/kg, n=18). Controls (n=18) were chosen and treated with 10 mL/kg paroline. Rats were sacrificed 24, 48 and 72 h after the treatment, and the blood, brain and sciatic nerves of these rats were prepared, respectively; the level of serum myelin basic protein (MBP) was detected and the morphological structure of myelin was observed with transmission electron microscope. Results The content of serum MBP in the 2 treatment groups was advanced markedly as compared with that in the control group 24, 48 and 72 h after the treatment (P<0.05);however, no significant difference was noted between the 2 treatment groups (P>0.05). The content of serum MBP was the highest 24 h after the treatment, gradually decreased following the prolongation of time; significant difference was noted between each 2 time points (P<0.05). Transmission electron microscope indicated abnormality in myelin of the central nerves at all time points, showing inordinate shape, rupture between layers and tangle among fibers; the abnormality was the most obvious 24 h after the treatment, gradually abatement following the prolongation of time; decreased myelin in the sciatic nerves was showed only at 24 h after the treatment, while normal at other time points. Conclusion A certain degree of abnormality of myelin of the central and peripheral nervous systems in rats can be caused by borneol.     

急性脑外伤患者脑脊液髓鞘碱性蛋白水平与其损伤类型的关系

目的 探讨急性脑外伤患者脑脊液髓鞘碱性蛋白(CSF-MBP)水平与其损伤类型的关系。 方法 采用放射免疫测定法(RIA)对42例急性脑外伤患者的CSF-MBP进行测定,并测定26例非神经系统疾病患者作对照。结果 严重脑内原发性损伤患者CSF-MBP平均水平显著高于轻中度脑内原发性损伤和单纯颅内血肿者(P<0.01),单纯颅内血肿患者CSF-MBP仅伤后第5天增高;弥漫性脑损伤组CSF-MBP水平显著高于局灶性脑损伤组(P<0.01),伤后1周内持续处于高水平状态。结论 CSF-MBP的测定有助于急性脑外伤患者损伤程度及类型的判定。     

Characterization of a novel monoclonal anti-myelin basic protein antibody: use in immunoblotting and immunohistochemical studies

Monoclonal antibodies (MAbs) to the myelin basic protein (MBP) were produced in CAF1 (BALB/c x A/J) mice immunized with intact bovine MBP. A number of MAbs were obtained, one of which was characterized in detail with respect to its isotype, antigenic determinant on the MBP, the spectrum of antigens with which it reacted in mouse brain, and its immunohistochemical staining characteristics. This monoclonal, GB-1 (an IgG1), recognized an epitope within residues 30-51 of bovine MBP. It also reacted with a family of MBP-related proteins present in brain homogenates of mice from 7-35 days. Immunohistochemically, GB-1 stained myelinated fibers and oligodendrocytes in the rodent CNS. A second monoclonal (GB-2, and IgM) was partially characterized. It reacted with intact MBP when it was immobilized to plastic or nitrocellulose, but it was not found to be useful for immunoblots or immunohistochemistry.     

Prominent white matter lesions develop in Mongolian gerbils treated with 100% normobaric oxygen after global brain ischemia

Summary Carotid arteries were occluded bilaterally for 15 min in two groups of Mongolian gerbils. The first group received 100% oxygen during the first 3 h of reperfusion. During that period, room air was given to the second group. After 3 h, both groups received room air. Brains of gerbils that died within 14 days after occlusion were removed, fixed in formalin and embedded in paraffin. Gerbils that survived 15–28 days were perfused with formalin before their brains were removed and embedded in paraffin. Adjacent, serially cut sections were stained with luxol fast blue (LFB)-H&E, cresyl violet, according to the Bodian method, or immunocytochemically with antisera raised against myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP). In brain sections of gerbils receiving 3 h of 100% oxygen, there were circumscribed white matter lesions in the corpus striatum, lateral thalamus, mesencephalon and posterior limb of the internal capsule. Myelin sheaths were swollen, fragmented and were less intensely stained by MBP antiserum. MBP and LFB-stained myelin fragments were present extracellularly and in macrophages. Many axons in these areas appeared undamaged. Previously described ischemic changes were found in gray matter and some areas of white matter in both groups. However, neurons in the deeper laminae of the cerebral cortex appeared to be better preserved in gerbils given oxygen. The results suggest that hyperoxia, if present immediately after transient brain ischemia, may damage myelin more severely than other cellular elements.     

小鼠实验性自身免疫性脑脊髓炎的病理变化

目的用髓鞘少突胶质细胞糖蛋白多肽(MOG35-55)诱发实验性自身免疫性脑脊髓炎(experi-m ental autoimmune encephalomyelitis,EAE)小鼠模型。方法应用MOG35-55抗原加完全弗氏佐剂免疫C57BL/6小鼠,利用光镜、电镜观察小鼠组织学改变。结果光镜下可见小血管周围炎细胞浸润,呈袖套状改变、血管周围明显脱髓鞘及神经元变性,B ieschowsky银染显示大量轴索肿胀和轴索卵形体的形成,电镜下可见髓鞘结构松散、断裂或融合,包括不同程度的髓鞘重建,脊髓病变广泛,程度重于脑部。结论EAE的病理改变为血管周围炎性细胞浸润、白质脱髓鞘及髓鞘重建。