LINGO-1抗体对多发性硬化模型小鼠髓鞘和认知功能的影响

发布时间：2018-05-20 20:25

本文选题：实验性过敏性脑脊髓炎 + 神经功能评分　；参考：《东南大学》2017年博士论文

【摘要】：研究背景:多发性硬化(multiple sclerosis, MS)是中枢神经系统中最常见的脱髓鞘疾病之一,好发于青壮年人,起病年龄在20～40岁。流行病学调查研究发现高达43%到70%的患者存在不同程度的认知功能障碍,并且患者的认知功能障碍随着时间越加严重。认知功能障碍严重影响了多发性硬化患者正常的生活和学习,给家庭和社会带来了沉重的负担。但是,目前对多发性硬化认知功能障碍的治疗手段有限,疗效欠佳,是临床上亟待解决的问题。尸检和影像学研究都证实了多发性硬化患者认知功能活动相关脑区存在明显的脱髓鞘损伤,并且研究还发现髓鞘损伤与认知功能障碍密切相关。然而,髓鞘损伤是否是多发性硬化认知功能障碍的治疗靶点还不清楚。含亮氨酸重复序列和免疫球蛋白结构域的Nogo受体的作用蛋白1(LRR and Ig domain containing, Nogo receptor interacting protein 1,Lingo-1)是一种特异性表达在中枢神经系统中具有抑制少突胶质细胞前体细胞(OPCs)分化成熟以及髓鞘化的蛋白,而Lingo-1的拮抗剂(Lingo-1抗体)具有促进髓鞘修复的作用。那么LINGO-1抗体是否能够用于治疗多发性硬化患者认知功能障碍目前尚不清楚。本研究中,我们检测了两种常见的多发性硬化脱髓鞘模型小鼠(实验性过敏性脑脊髓炎模型小鼠和Cuprizone诱导的脱髓鞘模型小鼠)的行为学,并用western blot和免疫荧光技术等观察了髓鞘蛋白和神经元轴突结构和功能蛋白表达水平以探讨:1、脱髓鞘模型小鼠是否存在认知功能障碍,以及小鼠认知功能相关脑区是否存在髓鞘损伤;2、LINGO-1抗体能否改善脱髓鞘模型小鼠的认知功能障碍及其病理损伤。最终,希望通过以上研究为多发性硬化认知功能障碍的治疗提供新思路,为LINGO-1抗体用于治疗多发性硬化认知功能障碍提供依据。第一部分:实验性过敏性脑脊髓炎小鼠的改良与评价目的:为了构建能够用于认知功能行为学检测的实验性过敏性脑脊髓炎(EAE)小鼠,对EAE造模药物的剂量进行了探索,并对改良的EAE模型进行了评价。方法:采用不同剂量的髓鞘少突胶质细胞糖蛋白(MOG35-55) (200μg MOG35-55/只、100μgMOG35-55/只、50μgMOG35-55/只、25μgMOG35-55/只)、灭活结核分枝杆菌(H37RA)(800 μg灭活结核分枝杆菌/只、250 μg灭活结核分枝杆菌/只、100 μg灭活结核分枝杆菌/只)和百日咳毒素(500 ng百日咳毒素/只、200 ng百日咳毒素/只)等药物进行EAE造模。造模后,按照神经功能评分标准进行神经功能评分;用开场试验检测小鼠自发活动能力。结果:与高剂量药物EAE造模组小鼠相比,低剂量药物造模组小鼠(25μgMOG35-55/只,100 μg灭活结核分枝杆菌/只,200 ng百日咳毒素/只),在病程中,每天的平均神经功能评分都明显降低。低剂量药物造模EAE组小鼠与阴性对照组相比,自发活动速度无明显差别[免疫后第16天,8.885 ±0.772 cm/s (EAE组),8.933±0.567 cm/s (阴性对照组);免疫后第 31 天,11.130±0.630 cm/s (EAE 组),10.670±0.959 cm/s (阴性对照组);免疫后第55 天,7.686±0.428 cm/s (EAE 组),8.313±0.918 cm/s (阴性对照组)](P0.05)。结论:低剂量药物(25μgMOG35-55/只,100μg灭活结核分枝杆菌/只,200ng百日咳毒素/只)所造的EAE模型小鼠存在轻度的神经功能障碍,但自发活动功能无明显损害。该模型可以用于研究多发性硬化认知功能障碍的病理机制和药物开发。第三部分:LINGO-1抗体促进髓鞘修复和EAE小鼠空间学习记忆功能恢复目的:探讨LINGO-1抗体能否改善实验性过敏性脑脊髓炎模型(EAE)小鼠的认知功能障碍(空间学习记忆障碍)及其病理损伤。方法:按照低剂量药物(25μgMOG35-55/只,100μg灭活结核分枝杆菌/只,200 ng百日咳毒素/只)诱导EAE小鼠模型;免疫后第14天开始系统的给予LINGO-1抗体治疗,剂量为10 mg/kg,每6天一次腹腔给药,给药直至行为学检测结束;免疫后第50天开始观察阴性对第二部分:实验性过敏性脑脊髓炎小鼠行为学和病理表现目的:探讨实验性过敏性脑脊髓炎(EAE)小鼠是否存在认知功能障碍(空间学习记忆障碍)和焦虑、抑郁样行为;探讨EAE小鼠认知功能相关的脑区是否存在脱髓鞘损伤。方法:按照低剂量药物(25μgMOG35-55/只,100μg灭活结核分枝杆菌/只,200 ng百日咳毒素/只)诱导EAE小鼠模型,分别在免疫后的早期(第13天)和免疫后的晚期(第55天)进行行为学检测,包括糖水偏爱实验、高架十字迷宫实验、旷野实验和水迷宫实验。在实验中,每只小鼠只完成其中一个时间点的所有行为学检测。行为学检测完成之后,提取脑组织,进行western blot检测。结果:EAE小鼠在其病程的早期和晚期,旷野实验、高架十字迷宫实验和糖水偏爱实验中,表现与阴性对照组相当(P0.05)。水迷宫实验中,EAE小鼠在病程的早期,表现与阴性对照组小鼠相当,不存在空间学习记忆障碍(P0.05);但是,EAE小鼠在病程的晚期出现明显的空间学习记忆障碍,表现为学习期EAE小鼠找到平台的时间长于对照组小鼠,尤其是学习期第一天(P0.05),检测期EAE小鼠目标象限运动路程百分比明显低于对照组的小鼠(P0.05)。Western blot检测结果显示EAE小鼠的海马旁回和穹窿-海马伞出现明显的脱髓鞘损伤,表现为髓鞘碱性蛋白(MBP)含量下降(P0.05)。结论:EAE小鼠在病程的晚期出现空间学习记忆障碍,这种空间学习记忆障碍可能与海马旁回和穹窿-海马伞髓鞘损伤有关。照组、EAE模型组及EAE模型LINGO-1抗体治疗组小鼠空间学习记忆能力和焦虑抑郁样行为;行为学检测结束后,提取脑组织,用western blot和免疫荧光等方法检测相应脑区的髓鞘相关蛋白,轴突结构和功能蛋白以及P13K/AKT/m-TOR信号通路上蛋白的表达。结果:LINGO-1抗体干预后,三组小鼠在糖水偏爱实验、旷野实验及高架十字迷宫实验中的表现没有显著差异(P0.05)。而Morris水迷宫实验中,在学习期和检测期EAE模型组小鼠的表现明显差于阴性对照组小鼠,与阴性对照组小鼠相比,学习期EAE小鼠站台潜伏期明显延长(P0.05),检测期站台周围活动路程明显减少(P0.05) ; LINGO-1抗体治疗组小鼠在水迷宫实验的表现优于EAE模型组,与EAE未治疗组相比,学习期站台潜伏期明显缩短(P0.05),检测期站台周围活动路程有所增加,其活动路程与阴性对照组相当(P0.05)。Western blot检测发现EAE小鼠的海马旁回和穹窿-海马伞出现明显的脱髓鞘损伤,表现为髓鞘碱性蛋白(MBP)的含量下降(P0.05),并伴有轴突顺向转运蛋白驱动蛋白轻链蛋白1(KLC1)含量的下降(P0.05)。给予具有重髓鞘功能的LIINGO-1抗体干预后,EAE小鼠海马旁回中的MBP和KLC1明显升高(P0.05),而穹窿-海马伞的MBP和KLC1无明显提高(P0.05)。此外,LINGO-1抗体治疗组小鼠海马旁回PI3K/AKT/m-TOR信号通路活化,表现为磷酸化的AKT和磷酸化的m-TOR较EAE未治疗组表达增加(P0.05)。结论:LINGO-1抗体可能通过修复髓鞘改善EAE小鼠的空间学习记忆障碍;LINGO-1抗体可能是通过活化PI3K/AKT/m-TOR信号通路来介导这一过程的;LINGO-1抗体治疗可能有助于脱髓鞘疾病患者认知功能的恢复。第四部分:LINGO-1抗体能够改善Cuprizone诱导脱髓鞘模型小鼠的病理损伤目的:探讨Cuprizone诱导脱髓鞘小鼠行为学改变以及背侧海马区的病理损伤;探讨LINGO-1抗体能否改善Cuprizone诱导脱髓鞘小鼠的行为学改变及其病理损伤方法:用0.2% (w/w) cuprizone喂养C57BL/6小鼠诱导脱髓鞘模型,每天喂养直至行为学检测结束;在造模后第三周开始进行LINGO-1抗体治疗,剂量为10mg/kg,每6天一次腹腔给药,给药直至行为学检测结束;造模后第9周开始观察正常对照组、cuprizone诱导脱髓鞘模型组、cuprizone诱导脱髓鞘模型LINGO-1抗体治疗组三组小鼠空间学习记忆能力和焦虑抑郁样行为;行为学检测结束后,提取脑组织,采用western blot和免疫荧光等方法检测背侧海马区的髓鞘相关蛋白、轴浆转运相关蛋白、轴突结构蛋白、突触相关蛋白、tau蛋白、和β淀粉样蛋白等的表达。结果:本研究发现Cuprizone诱导脱髓鞘模型小鼠存在轻度的空间学习能力障碍(P0.05),伴有背侧海马髓鞘碱性蛋白(MBP)含量下降(P0.05)。给予LINGO-1抗体干预后,Cuprizone诱导脱髓鞘小鼠记忆能力轻度提高,并伴有MBP含量增加(P0.05)。此外,在Cuprizone诱导脱髓鞘小鼠中,海马区同时有轴突功能蛋白顺向转运蛋白驱动蛋白1 (KLC1)、轴突结构相关蛋白神经丝蛋白轻链(NFL)和神经丝蛋白重链(NF200)含量显著下降(P0.05),LINGO-1抗体干预后,这些轴突功能蛋白和结构蛋白的水平得以提高(P0.05)。然而在Cuprizone诱导脱髓鞘模型小鼠的背侧海马中,不存在β淀粉样蛋白水平增加和tau蛋白的过度磷酸化(P0.05)。结论:Cuprizone诱导脱髓鞘模型小鼠背侧海马髓鞘损伤可能参与影响其空间学习障碍,LINGO-1抗体可能通过促进髓鞘修复来改善其空间学习障碍。髓鞘损伤可导致神经元轴突破坏,但是可能不会直接导致β淀粉样蛋白增加和tau蛋白过度磷酸化。重髓鞘可能是治疗多发性硬化认知功能障碍和恢复神经元功能的一种有效方式。
[Abstract]:Background: multiple sclerosis (MS) is one of the most common demyelinating diseases in the central nervous system, which is well distributed in young people with age of 20~40. Epidemiological studies have found that patients with up to 43% to 70% have different degrees of cognitive dysfunction and cognitive dysfunction with time. More serious. Cognitive dysfunction seriously affects the normal life and learning of patients with multiple sclerosis, which brings a heavy burden to the family and society. However, the treatment of multiple sclerosis is limited and the curative effect is not good. It is an urgent problem to be solved in clinical. Autopsy and imaging studies have confirmed the incidence of multiple sclerosis. There is an obvious demyelinating injury in the brain regions associated with cognitive functional activity in sclerosis, and the study has also found that myelin injury is closely related to cognitive dysfunction. However, it is not clear whether the myelin sheath injury is a target for the treatment of multiple sclerosis cognitive dysfunction. The Nogo receptor containing the repeating sequence of leucine and the immunoglobulin domain The protein 1 (LRR and Ig domain containing, Nogo receptor interacting protein 1, Lingo-1) is a specific expression in the central nervous system that inhibits the maturation and myelination of oligodendrocyte precursor cells (OPCs), and Lingo-1 antagonists have the role of promoting the repair of myelin sheath. It is not clear whether O-1 antibodies can be used in the treatment of cognitive dysfunction in patients with multiple sclerosis. In this study, we detected the behavior of two common multiple sclerosis demyelinating mice (experimental allergic encephalomyelitis model mice and Cuprizone induced demyelinating model mice), and used Western blot and immunofluorescence. The level of myelin protein and neuron axon structure and functional protein expression were observed by optical technology. 1, the presence of cognitive dysfunction in demyelinating model mice, and the presence of myelin injury in the brain regions of cognitive function in mice; 2, whether LINGO-1 antibody could improve the cognitive impairment and pathological damage in demyelinating model mice. Finally, we hope to provide new ideas for the treatment of multiple sclerosis cognitive dysfunction through the above research, and provide the basis for the use of LINGO-1 antibodies in the treatment of multiple sclerosis cognitive impairment. Experimental allergic encephalomyelitis (EAE) mice, the dose of EAE model drugs was explored, and the modified EAE model was evaluated. Methods: using different doses of myelin oligodendrocyte glycoprotein (MOG35-55) (200 mu g MOG35-55/, 100 mu gMOG35-55/ only, 50 u gMOG35-55/ only, 25 u gMOG35-55/), to inactivate Mycobacterium tuberculosis (H37). RA) (800 mu g inactivated Mycobacterium tuberculosis / only, 250 mu g inactivated Mycobacterium tuberculosis / only, 100 mu g inactivated Mycobacterium tuberculosis / only) and pertussis toxin (500 ng pertussis toxin / only, 200 ng pertussis toxin / only) to make EAE model. After making the model, the neural function score was evaluated according to the evaluation criteria of nerve function, and the opening test was used to detect the function. Results: compared with the high dose drug EAE model mice, the low dose drug model mice (25 mu gMOG35-55/ only, 100 micron g inactivated Mycobacterium tuberculosis / only 200 ng pertussis toxin / only), the average daily neural function score in the course of the disease was significantly reduced. The low dose drug model group EAE mice and the negative control group There was no significant difference in the speed of spontaneous activity [sixteenth days after immunization, 8.885 + 0.772 cm/s (EAE), 8.933 + 0.567 cm/s (negative control), thirty-first days after immunization, 11.130 + 0.630 cm/s (EAE group), 10.670 + 0.959 cm/s (negative control group); fifty-fifth days after immunization, 7.686 + 0.428 cm/s (EAE group), 8.313 + cm/s (negative control group)] (P0.05) Conclusion: low dose drugs (25 mu gMOG35-55/ only, 100 mu g inactivated Mycobacterium tuberculosis / only, 200ng pertussis toxin / only) have mild neurological dysfunction, but spontaneous activity has no obvious damage. This model can be used to study the pathological mechanism and drug development of multiple sclerosis recognition dysfunction. Third parts. LINGO-1 antibody promotes myelin repair and EAE mice's spatial learning and memory function recovery purpose: To explore whether LINGO-1 antibody can improve cognitive impairment (spatial learning and memory disorder) and pathological damage in experimental allergic encephalomyelitis model (EAE). Methods: to inactivate tuberculosis branches according to low dose of drug (25 u gMOG35-55/, 100 u G) The EAE mouse model was induced by bacilli / only 200 ng pertussis toxin and only fourteenth days after the immunization, the system was given the LINGO-1 antibody treatment, the dose was 10 mg/kg, every 6 days the intraperitoneal administration, the administration until the behavioral test ended, and the fiftieth days after the immunization, the negative to the experimental allergic encephalomyelitis mice behavior and disease. Objective: To investigate whether there is cognitive dysfunction (spatial learning and memory disorder) and anxiety and depressive behavior in experimental allergic encephalomyelitis (EAE) mice; to explore whether there is demyelinating injury in the brain area related to cognitive function of EAE mice. Methods: a low dose drug (25 u gMOG35-55/ only, 100 mu g inactivated Mycobacterium tuberculosis / only, 20 0 ng pertussis toxin / only induced EAE mice model, respectively, at early (thirteenth days) after immunization and late immunization (fifty-fifth days) to conduct behavioral tests, including sugar water preference experiment, elevated cross maze test, wilderness experiment and water maze test. In the experiment, each mouse only completed all the behavioral tests of one of the time points. After the behavior test was completed, the brain tissue was extracted and the Western blot was detected. Results: the EAE mice in the early and late stages of the disease, the open field experiment, the elevated cross maze experiment and the sugar water preference experiment, were similar to those of the negative control group (P0.05). In the water maze experiment, the EAE mice showed the equivalent of the negative control group in the early stage of the disease. There was no spatial learning and memory disorder (P0.05), but EAE mice had obvious spatial learning and memory disorders in the late course of the disease, which showed that the time of finding the platform in the learning period EAE mice was longer than that of the control group, especially the first day of the learning period (P0.05), and the percentage of the target quadrant movement of the EAE mice was significantly lower than that of the control group. The results of rat (P0.05).Western blot detection showed that the parachyhippocampal and fornix fornix parachute in the EAE mice had obvious demyelination damage, showing the decrease of myelin basic protein (MBP) content (P0.05). Conclusion: the EAE mice appeared in the late stage of the course of the spatial learning and memory disorder, which may be associated with the parahippocampal gyrus and the fornix hippocampus. The spatial learning and memory ability and anxiety and depressive behavior of mice in the EAE model group and the EAE model LINGO-1 antibody treatment group, the brain tissue were extracted after the end of the behavioral test, and Western blot and immunofluorescence were used to detect the myelin related egg white, the axon structure, the functional protein and the P13K/AKT/m-TOR in the corresponding brain region. The expression of protein on the signal pathway. Results: there was no significant difference between the three groups in the glucose water preference experiment, the open field experiment and the elevated cross maze test in the three groups (P0.05). In the Morris water maze experiment, the performance of the mice in the learning period and the detection period of the EAE model group was significantly worse than the negative control group, and the negative was negative. Compared with the group of mice, the incubation period of the EAE mice was obviously prolonged (P0.05), and the activity of the platform around the platform was significantly reduced (P0.05), and the mice in the LINGO-1 antibody treatment group were better than the EAE model group in the water maze test. Compared with the EAE group, the latency of the learning platform was significantly shortened (P0.05), and the activity around the platform platform was observed. The road distance increased, and its activity was similar to that of the negative control group (P0.05).Western blot detection found that the parachyhippocampal and fornix fornix parachute in the EAE mice had obvious demyelination damage, which showed a decrease in the content of myelin basic protein (MBP) (P0.05), accompanied by a decrease in the content of light chain protein 1 (KLC1) of the axon CIS transporter. P0.05). The MBP and KLC1 in the parahippocampal gyrus of EAE mice increased significantly (P0.05) in the parahippocampal gyrus (P0.05) in EAE mice, while the MBP and KLC1 in the fornix hippocampus were not significantly increased (P0.05). Furthermore, the PI3K/AKT/m-TOR signal pathway of the parahippocampal gyrus in the LINGO-1 antibody treatment group was activated by phosphorylated AKT and phosphorylation The expression of EAE untreated group increased (P0.05). Conclusion: LINGO-1 antibody may improve the spatial learning and memory impairment of EAE mice by repairing myelin sheath; LINGO-1 antibody may be mediated through the activation of PI3K/AKT/m-TOR signaling pathway; LINGO-1 antibody therapy may help to restore the cognitive function of demyelinating disease patients. The fourth part LINGO-1 antibody can improve the pathological damage of demyelinating model mice induced by Cuprizone: To explore the behavioral changes of demyelinating mice induced by Cuprizone and the pathological damage in the dorsal hippocampus, and to explore whether LINGO-1 antibody can improve the behavior modification and pathological damage of demyelinating mice induced by Cuprizone: 0.2% (w/w) cuprizone C57BL/6 mice were fed with demyelinating model and were fed every day until the end of behavioral test. Third weeks after the model, the LINGO-1 antibody was treated, the dose was 10mg/kg, the dose was given every 6 days, the drug was given to the end of the behavioral test, and the normal control group was observed at ninth weeks after the model, and the demyelinating model group was induced by cuprizone, cuprizone The three groups of mice were induced by demyelinating model LINGO-1 antibody in space learning and memory ability and anxiety and depressive behavior. After the end of the behavioral test, the brain tissue was extracted. Western blot and immunofluorescence were used to detect the myelin related protein in the dorsal hippocampus, axon transport phase protein, axon structure protein, synapse related protein, tau egg. Results: This study found that the Cuprizone induced demyelinating model mice had a mild spatial learning disability (P0.05), accompanied by a decrease in the content of myelin basic protein (MBP) in the dorsal hippocampus (P0.05). After the prognosis of LINGO-1 antibody, the memory ability of demyelinating mice induced by Cuprizone was slightly improved, accompanied by MBP. Content increased (P0.05). In addition, in Cuprizone induced demyelinating mice, the axon functional protein CIS transporter 1 (KLC1) was found in the hippocampus, and the axon structure related protein neurofilament light chain (NFL) and neurofilament heavy chain (NF200) content decreased significantly (P0.05). The prognosis of LINGO-1 antibody stem, these axon functional proteins and junctions The level of protein structure is improved (P0.05). However, there is no increase in beta amyloid protein level and over phosphorylation of tau protein (P0.05) in the dorsal hippocampus of demyelinating model mice induced by Cuprizone. Conclusion: Cuprizone induced demyelinating mouse dorsal hippocampus myelin injury may be involved in the influence of the spatial learning disability, LINGO-1 antibody. It may improve the spatial learning disorder by promoting myelin repair. Myelin injury may lead to neuronal axonal damage, but it may not directly lead to the increase of beta amyloid and tau protein overphosphorylation.
【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R744.51;R-332

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