【作者】 杨扬；

【导师】 吴卫平；

【作者基本信息】 中国人民解放军军医进修学院 ， 神经病学， 2010， 博士

【摘要】 目的：研究降低纤维蛋白对多发性硬化（MS）动物模型实验性自身免疫性脑脊髓炎（EAE）小鼠的预防及治疗作用,初步探讨纤维蛋白沉积在多发性硬化中的作用机制。方法：采用MOG_35-55免疫的C57BL/6雌性小鼠,诱发EAE动物模型,分别在免疫后立即（预防组）及免疫后出现症状（治疗组）隔日连续给予降纤药巴曲酶（30BU／kg）注射至实验结束,观察发病情况并进行临床症状评分。于免疫后30天、40天及60天分别将预防组、治疗组及EAE对照组小鼠脊髓与小脑组织取材后进行组织病理学与免疫组化染色分析,Western-blot和实时荧光定量PCR技术及体外]RAW264.7巨噬细胞培养观察药物干预对炎性浸润、脱髓鞘、纤维蛋白沉积及胶质细胞活化的影响。此外,还采用神经电生理技术观察小鼠视觉诱发电位（VEP）P100的潜伏期和波幅的变化。结果：降纤预防组及治疗组均可明显减轻EAE小鼠的发病症状、降低临床评分。巴曲酶预防组和治疗组较EAE未用药对照组小鼠炎性细胞浸润明显减少、髓鞘脱失及胶质细胞活化减轻,但轴索损害的改善作用不明显。免疫组化及免疫荧光双标结果表明,预防组和治疗组较EAE未用药对照组的小脑MBP表达升高而GFAP表达降低。Western-blot结果示MHC-I类分子蛋白表达无显著差异,p-Akt表达降低而MBP蛋白表达升高。实时荧光定量PCR技术也证实了巴曲酶预防及治疗组MBP的mRNA表达升高,t-PA的mRNA表达也升高。此外,纤维蛋白和LPS一样可诱导体外培养的RAW264.7巨噬细胞发生炎症反应,表现为细胞体积变大并有树突样改变,巴曲酶加入后可逆转这种巨噬细胞的树突样变化。同时,纤维蛋白免疫荧光检测,在预防组和治疗组阳性信号较EAE对照组降低,但通过检测小鼠眼静脉血的纤维蛋白原浓度发现,预防和治疗组有降低趋势,但与对照组相比差异无明显统计学意义。神经电生理技术观察结果提示,EAE组小鼠较空白对照组小鼠的闪光VEP的P100潜伏期延长、波幅减低,而巴曲酶用药组小鼠的P100潜伏期延长、波幅减低有所改善。结论：在EAE的发病过程中纤维蛋白沉积发挥了重要作用,巴曲酶通过有效降低EAE小鼠体内的纤维蛋白从多个方面改善临床症状和发病过程。目的研究血清AQP-4抗体对视神经脊髓炎的诊断价值。方法收集解放军总医院2008年11月至2009年6月住院和门诊临床诊断的患者共80例,其中NMO及高危NMO患者23例（NMO 11例,HR-NMO 12例）,经典MS患者36例,临床孤立综合征（clinical isolated syndrom, CIS）11例（单次发病的视神经炎2例,脊髓炎5例及颅内脱髓鞘4例）,其他神经系统疾病患者10例（包括亚急性联合变性1例、脑梗死5例、脑出血3例、神经纤维瘤1例）。采用细胞间接免疫荧光法检测所有患者血清AQP-4抗体滴度水平,比较各组患者临床特点及AQP-4水平。结果NMO、HR-NMO组与MS. CIS组相比,发生严重视神经炎、横贯性脊髓炎、有颅脑MRI正常表现、脊髓病灶超过3个椎体节段及血清AQP-4抗体阳性均有统计学意义（p＜0.05）.NMO与HR-NMO组血清AQP-4抗体水平显著高于MS组,而MS组血清AQP-4抗体又略高于CIS组与其他疾病组。血清AQP-4抗体对诊断NMO的灵敏度达82.6%,特异性达96.2%。结论血清AQP-4抗体的检测对视神经脊髓炎的诊断及鉴别诊断有一定的价值。目的研究中枢神经系统炎性脱髓鞘疾病患者血清AQP-4抗体、细胞因子干扰素-Y （IFN-γ）及白细胞介素-4 （IL-4）水平,以及AQP-4抗体与IFN-y和IL-4间的相关性,探讨视神经脊髓炎（NMO）和多发性硬化（MS）的发病机制。方法入选患者共130例,其中NMO患者41例,MS患者59例,临床孤立综合征（CIS）患者15例,其他神经系统疾病患者15例。采用细胞转染的间接免疫荧光法检测患者血清AQP-4抗体滴度,ELISA法检测患者血清IFN-y和IL-4水平,比较各组患者血清AQP-4抗体、IFN-y和IL-4水平并分析AQP-4抗体与IFN-y及IL-4的相关性。结果与MS组比较,NMO组患者女性更多见,平均发病年龄偏大,其血清AQP-4抗体水平显著高于MS组、CIS组及其他疾病组（p<0.05）,而IFN-y和IL-4水平与另3组比较差异无统计学意义（p＞0.05）；MS组血清AQP-4抗体水平与CIS组及其他疾病组比较差异均无统计学意义（p>0.05）。NMO组血清AQP-4抗体水平与IFN-y和IL-4呈正相关（r分别为0.36、0.35,p<0.05）,MS组血清AQP-4抗体水平与IFN-y及IL-4无明显相关性（r分别为0.03、-0.10,p>0.05）。结论血清AQP-4抗体可用于鉴别NMO和MS及其他炎性脱髓鞘病；NMO患者血清AQP-4抗体增高可能与细胞因子IFN-y和IL-4有一定关系。更多还原

【Abstract】 Objective Multiple sclerosis (MS) was characterized with widespread inflammatory demyelination and axonal loss of central nervous system (CNS). Fibrinogen (fibrin) deposition was considered as one of the pathogenesis of MS. In the present study, we explored the capacity of prophylactic and therapeutic potential of fibrin depletion by batroxobin to affect the inflammatory demyelination process in experimental autoimmune encephalomyelitis (EAE) mice model.Methods In MOG-induced EAE, batroxobin was separately injected after disease inducement immediately (prevention) and after the appearance of clinical manifestations (suppression). At the time of postimmunization(p.i.) day 30,40,60, spinal cords and cerebellum tissue samples from EAE mice were collected for histopathologic and immunohistochemical methods. Molecular biological methods such as Western-blot and Real-time PCR were supplied to detect MBP, p-Akt, MHC-I and t-PA. Meantime, RAW264.7 cells were cultured for immunofluorescence to obseve the activation of macrophages irritated by fibrinogen after supplement of batroxobin. In addition, the latency and amplitude of P100 in flash VEP of batroxobin-treated mice and EAE control mice were observed.Results Our study found that prevention and suppression with batroxobin significantly ameliorated clinical severity of EAE, reduced inflammatory cells infiltration, demyelination and suppressed the activation of astrocytes and macrophages comprising the CD11b+population. The expressions of p-Akt were downregulated, while those of MBP were upregulated in prevention and suppression with batroxobin mice as compared to control mice. Moreover, our results showed that in vitro adding of batroxobin reversed the dendric-like formation of macrophages irritated by fibrinogen under inflammatory conditions. Furthermore, the latency was slightly prolonged and amplitude decreased of P100 in flash VEP of batroxobin-treated mice than those of EAE control mice.Conclusions In conclusion, fibrinogen depleting agent batroxobin has a benefical effect on EAE, therefore, strategy targeting fibrin as a potential therapy for EAE maybe helpful for the treatment of MS patients. Objective To investigate the value of AQP-4 antibody in diagnosis of neuromyelitis optica (NMO).Methods A total of 80 patients with NMO, high-risk NMO(HR-NMO),multiple sclerosis (MS), clinical isolated syndrome(CIS) and other neurological diseases (subacute combined degeneration, cerebral infarction, intracranial hemorrhage and neurofibroma) were included in the study. The titres of all patients’ serum AQP-4 antibody were detected by indirect immunofluorence.Results There were significantly differences between patients with NMO, HR-NMO and MS, CIS, who experienced severe optical neuritis, transverse myelitis, brain normal in MRI scan, spinal-cord lesion more than 3 segments and serum AQP-4 antibody positive(p<0.05). The level of serum AQP-4 antibody of NMO, HR-NMO was higher than that of MS, while the level of serum AQP-4 antibody of MS was slightly higher than that of CIS and other diseases. The sensitivity and specificity of AQP-4 antibody were 82.6%,96.2%respectively in diagnosis of NMO.Conclusion AQP-4 antibody is valuable to diagnose NMO and differentiate from MS. Objective To investigate serum titres of AQP-4 antibody, levels of interferon-y (IFN-y) and interleukin-4 (IL-4) and their correlations in patients with neuromyelitis optica (NMO), multiple sclerosis (MS) and clinical isolated syndrome (CIS) in order to study the pathogenesis of NMO and MS.Methods A total of 130 patients with NMO including high-risk NMO (n=41), MS (n=59), CIS (n=15) and other neurological diseases (n=15) were included in the study. The titres of all patients’serum AQP-4 antibody were detected by indirect immunofluorence, and the levels of IFN-y and IL-4 were measured by ELISA kits.Results The titre of serum AQP-4 antibody in patients with NMO is much higher than those of patients with MS, CIS, and other diseases (p<0.05). However, there is also no significant difference of serum AQP-4 antibody among MS, CIS and other diseases(p>0.05). There is no significant difference of serum IFN-y and IL-4 among NMO, MS and other diseases(P>0.05). The titres of serum AQP-4 antibody in patients with NMO have positive correlation with IFN-y and IL-4 (r=0.36,0.35 respectively, p<0.05),on the other hand, the titres of serum AQP-4 antibody in patients with MS have not significant correlation with IFN-y and IL-4 (r=0.03,-0.10 respectively,p>0.05)。Conclusions AQP-4 antibody is not only valuable to differentiate NMO, MS and other diseases, but also may be have related to cytokines IFN-y and IL-4.更多还原