淫羊藿苷对实验性自身免疫性脑脊髓炎（EAE）小鼠星形胶质细胞及轴突的影响

发布时间：2018-04-27 00:27

本文选题：实验性自身免疫性脑脊髓炎 + 淫羊藿苷　；参考：《广东药科大学》2016年硕士论文

【摘要】：【目的】中枢神经系统白质脱髓鞘病变是多发性硬化(multiple sclerosis,MS)的主要病理特点,近年有关MS的星形胶质细胞异常及轴突损害才逐步受到重视。我们的前期研究证实淫羊藿苷(ICA)可通过影响炎性细胞因子、HPA轴途径调节炎性免疫机制而发挥对实验性自身免疫性脑脊髓炎(EAE)小鼠症状缓解及髓鞘保护作用。本研究拟在前期研究的基础上,继续采用以复发缓解型为特征的雌性C57BL/6 EAE小鼠模型为研究对象,通过观察治疗前后各组间小鼠体重、神经功能评分变化以及脑胼胝体切片GFAP(单标)、NF200+β-APP(双标)免疫荧光染色变化,探讨EAE小鼠脑星形胶质细胞异常和轴突损害,并研究ICA对其影响,揭示ICA对EAE是否具有非炎性神经保护作用。【方法】第一部分选取SPF级65只7-9周龄C57BL/6雌性小鼠,15只作为正常对照组,50只用于造模。将小鼠麻醉后将油包水状态的MOG35-55抗原乳剂按每只小鼠300ug的剂量皮下注射接种于小鼠脊柱两侧皮下(分四个部位注射),在接种后当天及48小时后腹腔注射500ng百日咳毒素,初次免疫后第7天再次加强免疫1次。免疫后动态观察小鼠是否出现神经功能症状并对其神经功能损害进行评分。在小鼠疾病高峰期随机抽取3只EAE小鼠及3只正常小鼠处死,取脊髓腰膨大用于HE染色,光镜下观察脊髓组织学变化,确定造模是否成功。第二部分将造模成功后的EAE小鼠按评分相当的原则随机分配到3组内,分别为雌激素组(阳性对照组)、ICA组、模型对照组,加上正常对照组(空白对照组),共4组,分别给予己烯雌酚0.2mg/kg.d、ICA300mg/kg.d、等容量0.5%羧甲基纤维素钠、等容量0.5%羧甲基纤维素钠灌胃,连续给药5天,给药后第6天取材,以多聚甲醛心脏灌注取脑组织,经后固定,蔗糖脱水后进行冰冻切片分别进行荧光染色:(1)GFAP(星形胶质细胞标志)荧光单标染色:根据免疫荧光染色步骤分别加入一抗GFAP,带红色荧光的二抗Cy3并封片,在荧光显微镜下,GFAP发红色荧光;(2)NF200(轴突标志)+β-APP(轴突损害标志)荧光双标染色:根据免疫荧光染色步骤加入一抗NF200、β-AP P,分别加入带绿色荧光的二抗Alexa Fluor488、带红色荧光的二抗Cy3并封片,在荧光显微镜下,NF200发绿色荧光,β-APP发红色荧光。将以上染色后的切片在40×物镜荧光显微镜下观察并拍摄照片。采用Image J软件对GFAP、NF200+β-APP图片进行荧光强度分析,采用SPSS Statistics 17软件行统计学分析,应用Graph Pad Prism软件进行作图。计量资料采用`x±s表示。多组间比较采用单因素方差分析,P0.05为差异有统计学意义,P0.01为差异有显著统计学意义。相关趋势变量采用Pearson相关分析。【结果】第一部分小鼠于免疫后第13-15天开始发病,2-3天后达疾病高峰,以尾部无力为首发症状的小鼠占大部分,亦有少部分小鼠以行走时平衡障碍发病,逐渐加重至肢体无力、肢体瘫痪。最高神经功能评分达5分,正常对照组小鼠无明显异常。对EAE小鼠脊髓切片HE染色可见大量淋巴细胞浸润,以血管周围显著,呈“袖套样”改变。而正常对照组未见病变。根据小鼠症状、神经功能评分及HE染色组织学改变说明构建EAE小鼠模型成功。第二部分(1)小鼠体重变化:小鼠接受抗原刺激后,体重开始出现下降,随着时间的推移,体重下降越来越明显。给予治疗后,雌激素组和ICA组EAE小鼠体重有明显增加(P0.01),治疗后各组与模型对照组比较,雌激素组和ICA组体重有明显改善(P0.01),雌激素组与ICA组比较无明显差异(P0.05)。(2)小鼠神经功能变化:雌激素组和ICA组EAE小鼠治疗后神经功能评分有明显改善(P0.05),治疗后各组与模型对照组比较,雌激素组和ICA组神经功能评分亦有明显好转(P0.05),雌激素组与ICA组比较无明显差异(P0.05)。(3)EAE小鼠存在星形胶质细胞异常及轴突损害:模型对照组GFAP(星形胶质细胞标志)荧光强度比正常组显著增高,同时细胞形态异常,数量增加,说明EAE小鼠存在星形胶质细胞活化增殖;模型对照组轴突(NF200标记)密度较正常组明显下降,说明EAE小鼠存在轴突损害。(4)ICA对EAE小鼠星形胶质细胞的影响:正常组的星形胶质细胞细胞体较小,突起如细丝状向周边延伸,形态未见异常,GFAP(星形胶质细胞标志)荧光强度低;模型对照组荧光强度比正常组显著增高(P0.05),且其处于活化状态,细胞形态异常,胞体增大且变得不规则,突起粗短;与模型对照组相比,雌激素组及ICA组荧光强度明显降低(P0.05),形态基本接近正常。雌激素组与ICA组比较无明显差异(P0.05)。(5)ICA对EAE小鼠轴突的影响:模型对照组轴突(NF200标记)密度较正常组明显下降(P0.01);雌激素、ICA治疗组轴突密度较模型对照组明显增高,均具有统计学意义,其中ICA组增高更明显(P0.01),雌激素组与ICA组之间未见明显差异(P0.05)。而在正常组未见损害轴突(β-APP标记),模型对照组β-APP荧光强度最高,表明轴突损害最严重,与模型对照组比较,经治疗后雌激素组和ICA组β-APP荧光强度均有明显下降(P0.01),雌激素组与ICA组之间未见明显差异(P0.05)。【结论】(1)EAE小鼠胼胝体内存在星形胶质细胞异常,表现为增殖活化状态,细胞形态异常,数量增加;且同时存在轴突损害,表现为轴突密度下降;(2)ICA治疗后,EAE小鼠胼胝体内星形胶质细胞活化减少,ICA可能通过抑制星形胶质细胞活化增殖来改善模型鼠神经功能损害;(3)ICA对EAE小鼠具有非炎性神经保护作用,可能与其调节轴突再生,减少轴突损害密切相关。
[Abstract]:[Objective] the central nervous system white matter demyelination is the main pathological feature of multiple sclerosis (MS). In recent years, the astrocyte abnormalities and axonal damage of MS have been gradually paid attention to. Our previous study confirmed that icariin (ICA) can affect inflammatory cytokines, and HPA axis pathway regulates inflammatory immunity. On the basis of the previous study, this study intends to continue to use the female C57BL/6 EAE mouse model characterized by remission type on the basis of the previous study. By observing the weight of mice and the changes of nerve function score in each group before and after the treatment. And the changes of GFAP (Dan Biao) and NF200+ beta -APP (double standard) immunofluorescence staining of the corpus callosum, explore the abnormalities of astrocyte and axon damage in the brain of EAE mice, and study the effect of ICA on it, and reveal whether ICA has non inflammatory neuroprotective effect on EAE. [Methods] the first part selected 65 7-9 weeks old C57BL/6 female mice at SPF level and 15 mice. For the normal control group, 50 rats were used to build the model. After the mice were anesthetized, the MOG35-55 antigen emulsion was injected subcutaneously on the two sides of the spinal column at the dose of 300ug in each mouse (four parts of the mice). The peritoneal injection of 500ng 100 day cough toxin was intraperitoneally injected after the day of inoculation and 48 hours after the inoculation, and the immunization was strengthened again after the first immunization. The 1 time. After immunization, the neurological function symptoms were observed and the neurological impairment was evaluated. 3 EAE mice and 3 normal mice were killed at the peak period of the disease. The spinal lumbar enlargement was used for HE staining. The histological changes of the spinal cord were observed under the light microscope to determine whether the model was successful. The second part would succeed in making the model. After the EAE mice were randomly assigned to 3 groups according to the principle of score, they were estrogen group (positive control group), group ICA, model control group, plus normal control group (blank control group), total of 4 groups, respectively given diethylstilbestrol 0.2mg/kg.d, ICA300mg/kg.d, 0.5% carboxymethyl cellulose sodium, equal capacity of 0.5% carboxymethyl cellulose sodium gavage. After 5 days of administration, the brain tissue was taken for sixth days after administration, and the brain tissue was perfused with paraformaldehyde at the heart. After being immobilized, the frozen sections were stained respectively after the sucrose was dehydrated: (1) GFAP (astrocyte marker) fluorescence single mark staining: according to the immunofluorescence staining steps, a anti GFAP, two anti Cy3 with red fluorescence and the fluorescence were added to the fluorescence. Under the microscope, GFAP hair red fluorescence; (2) NF200 (axon sign) + beta -APP (axon damage marker) fluorescent double labeling staining: according to immunofluorescence staining steps added to a anti NF200, beta -AP P, adding green fluorescence of the two anti Alexa Fluor488, red fluorescent two anti Cy3 and seal, under the fluorescence microscope, NF200 hair green fluorescence, beta -APP hair Red fluorescence. The above stained slices were observed and photographed under the 40 * objective microscope fluorescence microscope. Image J software was used to analyze the fluorescence intensity of GFAP and NF200+ beta -APP images. The SPSS Statistics 17 software was used for statistical analysis and Graph Pad Prism software was used for mapping. Using single factor analysis of variance, P0.05 was statistically significant and P0.01 had significant statistical significance. The correlation trend variable adopted Pearson correlation analysis. [results] the first part of the first part of the mice began to attack on the 13-15 day after the immunization, and reached the peak of the disease in 2-3 days. The mice were divided by walking balance disorder, and gradually increased to limb weakness and limb paralysis. The maximum nerve function score was 5 points. There was no obvious abnormality in the normal control group. A large number of lymphocyte infiltration was observed in the spinal section of EAE mice with HE staining, and the "sleeve like" changes were observed around the blood vessels, but the normal control group had no pathological changes. The mice symptoms, the neurological function score and the HE staining histological changes showed that the model of the EAE mice was successful. Second (1) the body weight changes in mice: the weight of mice began to decrease after receiving the antigen stimulation, and the weight loss became more and more obvious as time went on. After treatment, the weight of EAE mice in the estrogen group and the ICA group increased significantly (P0. 01), compared with the model control group, the weight of estrogen group and ICA group was significantly improved (P0.01), and there was no significant difference between the estrogen group and the ICA group (P0.05). (2) the changes of neural function in the mice were significantly improved (P0.05) after the treatment of the estrogen group and the ICA group of EAE mice (P0.05). The female group was compared with the model control group after treatment. There was a significant improvement in nerve function score in both group and ICA group (P0.05). There was no significant difference between the estrogen group and the ICA group (P0.05). (3) there was astrocyte abnormality and axon damage in EAE mice: the fluorescence intensity of GFAP (astrocyte marker) in the model control group was significantly higher than that in the normal group, and the cell morphology was abnormal and the number was increased, indicating that the EAE was small. The rat astrocytes were activated and proliferated; the density of the axon (NF200 markers) in the model control group was significantly lower than that in the normal group, indicating the axon damage in the EAE mice. (4) the effect of ICA on astrocytes in EAE mice: the astrocytes in the normal group were smaller, and the protuberances extended like the filaments to the periphery, and the morphology was not abnormal, GFAP (Star gum) The fluorescence intensity of the stromal cells was low, and the fluorescence intensity of the model control group was significantly higher than that of the normal group (P0.05), and it was in the activation state, the cell morphology was abnormal, the cell body increased and became irregular and the protuberance was short. Compared with the model control group, the fluorescence intensity of the estrogen group and the ICA group decreased significantly (P0.05), and the morphology was almost normal. Estrogen group and IC were almost normal. There was no significant difference in the A group (P0.05). (5) the effect of ICA on the axon of EAE mice: the density of the axon (NF200 marker) in the model control group was significantly lower than that in the normal group (P0.01). The axon density in the estrogen and ICA treatment group was significantly higher than that in the model control group, and all of them were statistically significant, and the increase of ICA group was more obvious (P0.01), and no between the estrogen group and the ICA group. There was no significant difference (P0.05) in the normal group (beta -APP). The fluorescence intensity of beta -APP in the model control group was the highest, indicating that the axon damage was the most serious. Compared with the model control group, the fluorescence intensity of the beta -APP in the estrogen group and the ICA group decreased significantly after treatment (P0.01), and there was no significant difference between the estrogen group and the ICA group (P0.05). (1) there was an abnormal astrocyte in the corpus callosum of EAE mice, which showed proliferation activation state, abnormal cell morphology, increasing number, and axon damage, showing a decrease in axon density. (2) after ICA treatment, the activation of astrocytes in the corpus callosum of EAE mice decreased, and ICA could inhibit the proliferation of astrocytes by inhibiting astrocytes. (3) ICA has a non inflammatory neuroprotective effect on EAE mice, which may be related to the regulation of axonal regeneration and axonal damage.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R277.7

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