氟西汀对实验性自身免疫性脑脊髓炎小鼠LCN2及CXCL10的影响

发布时间：2018-01-24 19:56

本文关键词： 实验性自身免疫性脑脊髓炎 LCN2 CXCL10 多发性硬化氟西汀　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景多发性硬化（multiple sclerosis,MS）是中枢神经系统（central nervous system,CNS）炎性脱髓鞘疾病，病因和发病机制尚不明确。近年来其发病呈现明显的增高趋势，是导致青年人神经功能障碍的主要疾病,严重威胁着青年人的健康和生存质量。载脂蛋白2（Lipocalin2,LCN2）作为新发现存在于脑脊液和脑实质的蛋白质，在炎症、免疫反应中起到重要作用。在MS患者脑脊液可以检测到增高的LCN2水平，提示载脂蛋白2水平可能与MS有关联，包括成为MS脑脊液中的标记物。随着科技日新月异,涉及中枢神经系统脱髓鞘疾病的诊断方法越来越精细，从而能够更好的服务临床。但是，当前更多的MS患者通过用药仅能缓解病情，所以，MS的病理生理机制需要进行进一步深入研究，从而为多发性硬化的治疗提供更多理论及实验依据。目的通过髓鞘少突胶质细胞糖蛋白（myelin oligodendrocyteglycoprotein35-55,MOG35-55)免疫小鼠构建经典的实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis，EAE）模型，测定小鼠脑组织中LCN2表达情况及其血清中CXCL10水平，以及使用氟西汀后的变化情况，探讨氟西汀在MS疾病中的作用机制。方法 1.实验分组和干预方法：选取雌性C57BL/6小鼠总共80只，8-10周龄大小，体重在18～20g，被随机分为四组，每组20只。用生理盐水加完全弗氏佐剂（Complete Freund'sAdjuvant，CFA）免疫动物对照组小鼠。以MOG35-55及含有卡介苗的CFA免疫动物EAE组小鼠，即建立EAE模型。干预组建模前经紫外线（280～320nm）照射，以MOG35-55及含有卡介苗的CFA免疫动物，建立EAE模型。氟西汀组建模前经紫外线（280～320nm）照射，以MOG35-55及含有卡介苗的CFA免疫动物，建立EAE模型，免疫当天开始给予氟西汀（10mg/kg）灌胃，直至处死。对照组、干预组和EAE组小鼠均每天给予等量生理盐水灌胃处理，并持续至处死。 2.构建动物模型方法：制作诱导乳剂是通过向MOG35-55中加入生理盐水进行稀释，稀释浓度为3mg/ml，再将此液体与等体积的完全弗氏佐剂混合，使用玻璃注射器最终抽吸成油包水状。按0.2ml/只给予模型组及干预组C57BL/6小鼠背部中央偏头侧皮下4点注射诱导乳剂，于免疫后第0天、第2天分别进行腹腔注射百日咳毒素400ng，建立EAE动物模型。 3.神经功能评分：免疫结束后，每天观察小鼠精神状态，进食以及活动情况等有无变化。同时评估小鼠神经功能，采用国际认可的Kono评分标准。 4.检测指标：取额叶及脑室周围脑组织做石蜡切块，行HE染色，用免疫组化化学法测定脑组织中LCN2的表达。ELISA试剂盒检测血清CXCL10含量。结果 1.C57BL/6小鼠发病情况：小鼠经抗原免疫后临床症状逐渐加重，可表现为精神萎靡，食欲差，体重下降，皮毛欠光滑等情况，并且出现尾部无力，继而后肢无力，最后前肢无力甚至大小便均失禁等临床症状，小鼠发病临床表现呈现动态性改变，以后稳步进入慢性期阶段。干预组、氟西汀组两组小鼠发病时间和病情高峰期均较EAE组出现后移，临床症状也相对减轻。 2.HE染色结果：EAE组小鼠脑组织伴有明显的炎性细胞浸润现象，在脑组织病变主要集中在软膜下和脑室周围白质，可见血管袖套现象和明显的脑膜炎表现，以单核细胞浸润为主；干预组、氟西汀组中炎性细胞浸润现象明显减少。 3.免疫组化染色结果：各组均有阳性细胞存在，EAE组白质空泡形成、疏松明显。EAE组（57.33±3.670）、氟西汀组（48.00±5.899）和干预组（49.33±5.785）阳性细胞数平均秩次明显高于对照组（13.00±3.742），且具有统计学意义（P＜0.05）。氟西汀组和干预组阳性细胞数较EAE组明显减少（P＜0.05）。氟西汀组阳性细胞数较干预组无明显变化（P＞0.05）。 4.小鼠血清CXCL10水平ELISA结果：EAE组CXCL10含量较氟西汀组高，，氟西汀组较对照组也升高（P＜0.05）。 5.Pearson相关分析显示：EAE组小鼠脑组织中LCN2阳性细胞数平均秩次和血清CXCL10含量之间存在正相关（P＜0.05）。结论 1.氟西汀可以推迟EAE小鼠的发病时间，减轻疾病对神经系统功能损害。 2.氟西汀能够减轻EAE小鼠脑组织中炎性细胞浸润程度，对EAE小鼠起到保护作用。 3.氟西汀能够降低EAE小鼠脑组织中LCN2表达，降低EAE小鼠血清CXCL10水平，发挥抗炎作用，提示其在治疗人类MS方面具有很有希望的应用前景。
[Abstract]:Research background
Multiple sclerosis (multiple sclerosis MS) is the central nervous system (central nervous system, CNS) in inflammatory demyelinating diseases, etiology and pathogenesis is still not clear. In recent years the incidence showed a significant increasing trend, is the leading cause of disease of young people nerve dysfunction, a serious threat to the health and survival of young people 2. The quality of apolipoprotein (Lipocalin2, LCN2) as a new found in cerebrospinal fluid and brain protein in inflammation, plays an important role in the immune response. MS can be detected in cerebrospinal fluid of patients with elevated levels of LCN2, suggesting that apo 2 levels may be associated with MS, including a marker of MS in cerebrospinal fluid the diagnosis method and with the science and technology change rapidly, involving the demyelinating disease of the central nervous system more precise, so as to better service in clinic. However, the more MS patients through medication can alleviate disease Therefore, the pathophysiological mechanism of MS needs to be further studied in order to provide more theoretical and experimental basis for the treatment of multiple sclerosis.
objective
The myelin oligodendrocyte glycoprotein (myelin, oligodendrocyteglycoprotein35-55, MOG35-55) in mice immunized with classical construction of experimental autoimmune encephalomyelitis (experimental autoimmune, encephalomyelitis, EAE) model, CXCL10 level and serum LCN2 expression in mouse brain were measured, and the change after fluoxetine, explore the mechanism of action of fluoxetine in MS disease in.
Method
1. experimental groups and intervention methods: a total of 80 female C57BL/6 mice were selected, aged 8-10 weeks, weighing 18 ~ 20g, were randomly divided into four groups, 20 rats in each group. Saline with complete Freund's adjuvant (Complete, Freund'sAdjuvant, CFA) immune animal control mice. CFA EAE mice with immune animal MOG35-55 and contain BCG, namely the establishment of EAE model. The intervention group before modeling by UV irradiation (280 ~ 320nm), CFA and MOG35-55 in animal immunity containing BCG, establish the EAE model. With the UV fluoxetine group (280 ~ 320nm) irradiation, CFA and MOG35-55 in animal immunity containing BCG, the establishment of EAE model of immune day given fluoxetine (10mg/kg) orally, until death. The control group, intervention group and EAE group were given normal saline treatment, and continued until death.
2. methods: the animal model induced by emulsion is diluted by adding normal saline to MOG35-55, dilution concentration is 3mg/ml, then the mixture of complete Freund's adjuvant and the liquid volume, the use of glass syringe aspiration into the final water in oil. According to 0.2ml/ only give the model group and intervention group C57BL/6 mice central 4 migraine lateral subcutaneous injection induced emulsion, on the zeroth day after immunization, second days were given intraperitoneal injection of pertussis toxin 400ng, an animal model of EAE was established.
3. nerve function score: after the end of immunization, we observed the changes of mental state, feeding and activity of mice daily. At the same time, we evaluated the neurological function of mice, and adopted the internationally recognized Kono scoring standard.
4. test index: take frontal lobe and periventricular brain tissues as paraffin blocks, perform HE staining, detect the expression of LCN2 in brain tissue by immunohistochemical method, and detect CXCL10 content in serum by.ELISA kit.
Result
The incidence of 1.C57BL/6 in mice: mice were immunized after clinical symptoms gradually worsened, can be manifested as listlessness, poor appetite, weight loss, less smooth fur and so on, and then the tail weakness, hind limb weakness, finally forelimb weakness or even urine will incontinence and other symptoms of onset clinical presentation of dynamic change, after steadily into the chronic phase stage. The intervention group, fluoxetine group two incidence and severity of the peak time of mice were higher than those in group EAE after the shift, the clinical symptoms are relatively reduced.
Results: 2.HE staining of brain tissue of mice in EAE group with significant infiltration of inflammatory cells in the brain lesions, mainly concentrated in the surrounding subpial and periventricular white matter, visible vascular cuff phenomenon and obvious manifestations of meningitis, infiltration of monocytes; the intervention group significantly reduced the infiltration of inflammatory cells in the fluoxetine group.
3. immunohistochemical staining results: there were positive cells were found in EAE group, the white matter vacuolization, osteoporosis group.EAE was (57.33 + 3.670), fluoxetine group (48 + 5.899) and intervention group (49.33 + 5.785) positive cell number and mean rank was significantly higher than the control group (13 + 3.742), and statistically the significance (P < 0.05). Fluoxetine group and intervention group the number of positive cells was significantly reduced compared with EAE group (P < 0.05). The number of positive cells compared with fluoxetine group the intervention group had no significant change (P > 0.05).
The results of CXCL10 level ELISA in serum of 4. mice: the content of CXCL10 in group EAE was higher than that in fluoxetine group, and in fluoxetine group was also higher than that of control group (P < 0.05).
5.Pearson correlation analysis showed that there was a positive correlation between the average number of LCN2 positive cells in the brain tissue of group EAE and the content of serum CXCL10 (P < 0.05).
conclusion
1. fluoxetine can postpone the onset time of EAE mice and reduce the damage to the nervous system.
2. fluoxetine can reduce the degree of inflammatory cell infiltration in the brain tissue of EAE mice and protect EAE mice.
3. fluoxetine can reduce the expression of LCN2 in the brain tissue of EAE mice, reduce the level of CXCL10 in serum of EAE mice, and play an anti-inflammatory role, suggesting that it has a very promising application prospect in the treatment of human MS.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.51

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