【作者】 张会朵；

【导师】 檀国军；

【作者基本信息】 河北医科大学 ， 神经病学， 2010， 硕士

【摘要】 目的:多发性硬化(multiple sclerosis,MS)是一种以中枢神经系统( central nerval system, CNS)白质脱髓鞘为主要病理特点的自身免疫性疾病。实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)是以特异性致敏的CD4+ T细胞介导为主的,中枢神经系统内小血管周围单个核细胞浸润及髓鞘脱失为特征的自身免疫性疾病,在发病机制和病变表征等方面与MS极为相似,作为一种实验动物模型被广泛用于研究MS。已有人报道在脑及脊髓损伤情况下,神经干细胞标志物表达增多,但关于神经干细胞在EA E大鼠发病、缓解、复发过程中脊髓中表达情况未见报道。在MS的发病机制中,氧化应激起着非常重要的作用。α-硫辛酸作为一种很强的天然抗氧化剂,已引起人们的广泛重视。它主要通过清除自由基,螯合金属离子,再生其他抗氧化剂而发挥其抗氧化作用。但目前关于α-硫辛酸对MS的研究很少,我们研究发现α-硫辛酸可以显著降低其发病率,减轻病情,其作用是否与促进神经干细胞增殖有关尚不清楚。本实验旨在通过对EAE大鼠发病过程中脊髓中神经干细胞相关指标(巢蛋白Nestin及增殖细胞核抗原PCNA)的测定及分析,了解MS发病过程中神经干细胞的增殖情况及意义。并通过α-硫辛酸对EAE大鼠的干预来观察神经干细胞相关指标的变化,了解α-硫辛酸对神经干细胞增殖的影响,从而为临床治疗MS提供新的思路和实验依据。方法:1动物分组:将36只成年健康雌性Wistar大鼠,体重180～200g,随机分出正常对照组6只,其余动物用新鲜豚鼠全脊髓匀浆(GPSCH)诱导免疫,待动物第一次发病后随机选出发病组大鼠6只,其余平均分为EAE组和硫辛酸组(LA组,100mg .kg-1 .d-1),每组再按病程分为缓解、复发两个亚组,每亚组6只,硫辛酸组动物没复发者,按预实验中统计的EAE组动物缓解复发平均天数分为干预后7天组(缓解期)和干预后14天组(复发期)。2模型制备:将新鲜豚鼠全脊髓制成匀浆,加上完全福氏佐剂(complete Freund′s adjuvant,CFA),佐剂中含卡介苗6mg/ml,经过乳化后按0.5ml/只分别于大鼠四肢足垫和背部皮下注射。硫辛酸组于动物发病后(神经功能评分达2分)第一天起给予相应剂量腹腔注射,连续注射7天(100mg .kg-1. d-1)。3神经功能评分:于免疫诱导当日及免疫后每天对动物的体重变化、精神状态、活动情况进行观察,并对动物进行神经功能评分,采用国际通用的Kono评分标准,分为5分:0分,正常;1分,动物尾部无力;2分,尾部无力+肢体无力;3分,肢体轻度麻痹;4分,肢体严重麻痹,被动翻身后不能复原;5分,濒死状态或死亡。EAE大鼠以神经功能评分达2分或以上者为动物模型成功的标准。4免疫组织化学染色:在动物麻醉状态下,快速取其脊髓中腰膨大处组织,4%多聚甲醛固定,石蜡包埋,组织切片,分别加Nestin一抗及PCNA一抗,经二抗,三抗孵育,DAB显色,苏木精复染,脱水透明,中性树胶封片,光学显微镜下观察Nestin及PCNA表达情况。结果:1脊髓Nestin表达:发病期阳性细胞数目最多,缓解期减少,复发期阳性细胞再次增多,分布较分散,未明显集中于病灶区。复发期与发病期比较,阳性细胞数减少,有统计学意义(P<0.05)。缓解期LA组与EAE组比较,无统计学意义(P>0.05)。复发期LA组与EAE组比较,阳性细胞数减少,有统计学意义(P<0.05)。2脊髓PCNA表达:发病期阳性细胞数目最多,缓解期减少,复发期阳性细胞数再次增多,并主要集中在炎性细胞浸润的血管“袖套样”病变处,与病灶分布基本一致,其他部位弥漫分布。复发期与发病期比较,阳性细胞数减少,有统计学意义(P<0.05)。缓解期硫辛酸组与EAE组比较,无统计学意义(P>0.05)。复发期LA组与EAE组比较,阳性细胞数减少,有统计学意义(P<0.05)。结论:1硫辛酸能促进EAE大鼠病情缓解,但对内源性神经干细胞的增殖程度未见明显影响。2细胞增殖程度与EAE病情发展密切相关;硫辛酸能在一定程度上减少细胞增殖程度,可能与减轻炎症反应有关。更多还原

【Abstract】 Objective:Multiple sclerosis (MS) is an inflammatory demyelination disease of the central nervous system (CNS) that causes relapsing and progressive neurological impairment. Experimental autoimmune encephalomyelitis (EAE), an autoimmune disease, characterised by myelinolysis and perivascular cuffings, is an ideal animal model for MS.There has been some reports that the neural stem cell marker expression is increasing when the brain and spinal injury, but there is few reports about that in relapsing-remitting EAE rat.Oxidative stress plays an important role in the pathogenesis of MS. Alpha-lipoic acid has attracted wide attention as a strong natural anti-oxidants. It mainly plays its antioxidant activity through scavenging free radicals, chelating metal ions, and regenerating other antioxidants. We found that alpha-lipoic acid can significantly reduce the morbidity of EAE, but weather it can promote the neural stem cell proliferation or not is unclear.In the research, we want to know the neural stem cell proliferation and its significance in EAE rat by detecting the induction of Nestin and PCNA. We administrate alpha-lipoic acid to the EAE rat in order to know its impact about the neural stem cell proliferation, thus to provide new ideas and experimental evidence to the treatment of MS.Methods:A total of 36 adult healthy female Wistar rats weighing between 180g and 200g were divided randomly into three groups: normal control group(6 rats),other rats were immunized subcutaneously in the four foot pads and backside by fresh guinea pig spinal cord homogenate (GPSCH) and complete Freund′s adjuvant(CFA). After the first paroxysm, divided randomly out 6 rats as disease group, others divided into EAE group and alpha-lipoic acid (LA group, 100mg. Kg-1. d-1), and this two groups were divided into 7 day group and 14 day group, separately.Clinical signs of EAE were assessed the mean of twice daily by two observations. Scores were assigned on the basis of the following symptoms: 1. tail weakness; 2. tail weakness plus limb asthenia; 3. mild limb paralysis; 4. severe limb paralysis; 5. moribund/dead. LA group were injected intra- peritioneal resectively LA100mg.Kg-1.d-1. This treatment was started on the first day of EAE outbreak and continued daily for 7 days.Rats were sacrificed after anesthesia with intraperitoneal injection. Tissue of the spinal cord was fixed with 4% formalin, then the tissue was embedded in paraffin and sectioned at 6μm thickness. The sections were stained with immunehistochemistry staining of Nestin and PCNA. At last, the results were analyzed with microscopy.Results:1 Nestin expression in the spinal cord: The number of positive cells in EAE disease stage is increased. In the remitted stage, it is decreased. In relapsing stage, it is increased again. The number of positive cells in spinal cord of rats in EAE disease group is higher than Relapsing group (P<0.05). In the remitted stage, there is no statistical significance between LA group and EAE group (P>0.05). In the relapsed stage, the number of positive cells of LA group is lower than that of EAE group (P<0.05). In the study, we find that there are Nestin positive cells similar with neuron or astrocytes.2 PCNA expression in the spinal cord: The number of positive cells in EAE disease stage is increased. In the remitted stage, it is decreased. In relapsing stage, it is increased again. The number of positive cells in spinal cord of rats in EAE disease stage is more than that in Relapsing stage (P<0.05). In the remitted stage, there is no statistical significance between LA group and EAE group (P>0.05). In the relapsed stage, the number of positive cells of LA group is lower than that of EAE group (P<0.05). And the positive cells are mainly distributed in the department of inflammatory cell infiltration.Conclusions:1 alpha-lipoic acid can protect the EAE rats from the severity of the disease, but it has no significant effect on the endogenous neural stem cell proliferation.2 The degree of cell proliferation is closely related to the development of EAE disease; alpha-Lipoic acid can reduce cell proliferation by reducing the degree of inflammation.更多还原