大鼠反复轻度脑创伤后神经行为学及免疫反应的变化研究

发布时间：2018-05-01 18:14

  本文选题：反复轻度脑创伤 + 神经行为学　； 参考：《天津医科大学》2017年硕士论文

【摘要】：研究目的:脑创伤(traumatic brain injury,TBI)因其高致残、高致死的特点,严重危害着人类的健康,已成为全世界第三大疾病。近年来,一种与反复轻度脑创伤(repetitive mild traumatic brain injury,rmTBI)相关的神经退行性疾病——慢性创伤性脑病(chronic traumatic encephalopathy,CTE)得到了研究学者们越来越多的关注,该疾病常见于退伍士兵、橄榄球运动员、拳击选手等。现阶段,相关研究主要集中于CTE相关的rmTBI模型中行为学和病理学的研究,而CTE相关的rmTBI模型的免疫学研究尚未报道,属于目前科学研究的空白领域。本实验首先通过改进的神经功能评分(mNSS)和Morris水迷宫实验(MWM),证实rmTBI大鼠模型存在长时程的神经行为学异常和认知功能障碍,其次运用免疫荧光术和流式细胞术初步研究rmTBI大鼠模型中T细胞亚群(CD3~+T细胞、CD3~+CD4+T细胞、CD3~+CD8+T细胞)以及小胶质细胞表型(M1型小胶质细胞、M2型小胶质细胞)的变化规律,以了解CTE相关rmTBI大鼠模型中长时程免疫反应的变化规律,为进一步探索其免疫机制奠定实验基础,同时也为进一步探讨CTE相关的新颖免疫防治策略提供理论依据。研究方法:1.mNSS和MWM评价大鼠rmTBI伤后42d内的神经行为学功能和认知学习水平。2.应用流式细胞仪检测大鼠rmTBI伤后脑组织以及外周血中1d、3d、7d、14d、28d以及42d的T细胞亚群(主要集中在CD3~+T细胞、CD3~+CD4+T细胞、CD3~+CD8+T细胞)的变化规律。3.应用免疫荧光、流式细胞仪检测大鼠rmTBI伤后脑组织中7d、14d、28d及42d的小胶质细胞表型(M1型小胶质细胞、M2型小胶质细胞)的变化规律。结果:1.(1)mNSS表明,大鼠rmTBI伤后42d内存在明显的神经行为学异常。(2)MWM表明,大鼠rmTBI伤后存在长时程的认知功能障碍。2.(1)大鼠rmTBI伤后,存在长时程中枢免疫功能的异常,T细胞(CD3~+T细胞)百分率呈双峰型趋势,在7d和42d显著上升。T细胞中CD3~+CD4+T细胞的百分率在伤后7d上升,之后持续下降。T细胞中CD3~+CD8+T细胞的变化趋势恰与CD3~+CD4+T细胞的变化趋势相反。(2)外周免疫中,T细胞(CD3~+T细胞)的百分率在伤后7d、14d均有显著的上升,在伤后28d、42d均有显著的下降。T细胞中CD3~+CD4+T细胞的百分率在伤后持续下降。T细胞中CD3~+CD8+T细胞的百分率在伤后7d至28d逐渐上升,随后42d下降。3.(1)小胶质细胞(Iba-l阳性)在伤后7d和42d显著增加。(2)小胶质细胞(Iba-l阳性)中,CD86+/CD11b+M1型小胶质细胞在伤后42d表达显著增加,CD206+/CD11b+M2型小胶质细胞在伤后7d表达显著增加。结论:1.大鼠rmTBI伤后,存在长时程的神经行为学异常和认知功能障碍。2.大鼠rmTBI伤后,存在中枢和外周免疫功能的紊乱,其中,慢性期的外周免疫抑制可能对rmTBI神经预后产生影响。3.大鼠rmTBI伤后,小胶质细胞表型存在一定的变化规律,急性期以M2型小胶质细胞为主,慢性期以M1型小胶质细胞为主,M1/M2型小胶质细胞的差异表达可能对rmTBI的继发性损伤产生影响。
[Abstract]:Objective: traumatic brain injury-tibi (TBI) has become the third major disease in the world because of its characteristics of high disability and high mortality, which seriously endangers the health of human beings. In recent years, researchers have paid more and more attention to a neurodegenerative disease called chronic mild traumatic brain encephalopathy, a neurodegenerative disease associated with repeated mild traumatic brain injury-rmTBI, which is common among veterans and rugby players. A boxer, etc At present, the related studies are mainly focused on the behavior and pathology of rmTBI models related to CTE, while the immunological studies of rmTBI models related to CTE have not been reported, which is a blank field of current scientific research. In this study, we first demonstrated the existence of long term neurobehavioral abnormalities and cognitive dysfunction in rmTBI rat model by improved neural function score (mNSS) and Morris water maze test. Secondly, immunofluorescence and flow cytometry were used to study the changes of T cell subsets (CD3 ~ CD4 T cells, CD3 ~ CD8 T cells) and microglial phenotypes (M 1 microglia / M 2 microglia) in rmTBI rats. In order to understand the changes of long term immune response in CTE related rmTBI rat model, to lay the experimental foundation for further exploring its immune mechanism, and to provide the theoretical basis for further exploring the novel immunological prevention and treatment strategy related to CTE. Methods: 1. MNSS and MWM were used to evaluate the neurobehavioral function and cognitive learning level within 42 days after rmTBI injury in rats. The changes of T cell subsets (mainly CD3 ~ CD4 T cells) in brain tissue and peripheral blood of rats after rmTBI injury were detected by flow cytometry. Immunofluorescence and flow cytometry were used to detect the changes of microglial phenotype M 1 microglia type M 2 microglia in rat brain tissue after rmTBI injury for 14 d and 42 d. Results 1. 1 / 1 / 1 / 1 / mNSS showed that there were obvious neurobehavioral abnormalities within 42 days after rmTBI injury in rats. MNSS showed that there was a long term cognitive impairment after rmTBI injury in rats. 2. 1) after rmTBI injury, there was no significant difference between the two groups. The percentage of abnormal T cells with long term central immune function showed a bimodal trend. The percentage of CD3 ~ CD4 T cells in T cells increased significantly at 7 and 42 days after injury, and the percentage of CD3 ~ CD4 T cells in T cells increased 7 days after injury. The percentage of CD3 ~ CD8 T cells in peripheral immunity increased significantly on the 14th day after injury, and the change trend of CD3 ~ CD8 T cells was opposite to that of CD3 ~ CD4 T cells. The percentage of CD3 ~ CD4 T cells in T cells decreased continuously on the 28th day after injury. The percentage of CD3 ~ CD8 T cells in T cells increased gradually from 7 to 28 days after injury. The expression of CD86 / CD11b M1 type microglia was significantly increased on the 42nd day after injury, and the expression of CD206 / CD11b M2 microglia increased significantly on the 7th and 7th day after injury. Conclusion 1. There were long term neurobehavioral abnormalities and cognitive dysfunction after rmTBI injury in rats. There are central and peripheral immune dysfunction after rmTBI injury in rats. The peripheral immunosuppression in chronic phase may affect the prognosis of rmTBI nerve. 3. After rmTBI injury, microglial phenotypic changes were observed in rats. In acute phase, M2 microglia were dominant, and in chronic phase, M1 microglial cells were dominant. The differential expression of M1 / M2 microglia might have an effect on the secondary injury of rmTBI.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

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