脑内小胶质细胞表型在癫痫发生过程中的作用研究

发布时间：2018-05-31 05:57

本文选题：癫痫发生过程 + 小胶质细胞　；参考：《重庆医科大学》2017年博士论文

【摘要】：第一部分癫痫发生过程中脑内小胶质细胞表型的动态变化目的:探索小鼠在癫痫发生过程中的不同时期脑内小胶质细胞表型以及表型相关细胞因子的动态变化。方法:选用7-8周龄C57BL/6J小鼠,分为正常对照组和实验组。实验组采用匹鲁卡品腹腔注射诱导惊厥持续状态(Status epilepticus,SE),同时进行脑电监测对模型进行验证。采用FCM对SE后不同时期小胶质细胞表型的变化进行动态监测;酶联免疫标记分析(Enzyme-linked immunosorbent assay,ELISA)监测SE后不同表型小胶质细胞所分泌的相关细胞因子白介素-1β(Interleukin-1β,IL-1β)、白介素-4(Interleukin-4,IL-4)、白介素-10(Interleukin-10,IL-10)的变化情况。结果:(1)匹鲁卡品能够诱发C57BL/6J小鼠出现惊厥表现,并且SE后脑电图改变符合癫痫发生过程中的各时期脑电图变化。(2)小鼠脑内小胶质细胞表型比例在SE后癫痫发生的过程中动态变化,M1型小胶质细胞比例在SE后迅速升高,并维持在较高水平,直至SE后20天左右开始逐渐下降,随后降至接近对照水平;M2型小胶质细胞比例在SE后迅速下降,随后逐渐升高,在SE后第20天左右达到高峰,并于SE后第28天将至接近对照水平。(3)脑内M1型小胶质细胞分泌的典型细胞因子IL-1β的水平的波动趋势与M1型小胶质细胞比例的变化趋势基本一致;M2型小胶质细胞分泌的典型细胞因子IL-4、IL-10的水平与M2型小胶质细胞比例的变化趋势基本一致。结论:SE后癫痫发生过程中小胶质细胞的不同表型所占比例动态变化,表型相关细胞因子的水平与表型变化趋势基本一致,说明小胶质细胞的表型变化参与了癫痫发生过程。第二部分腹腔注射IFN-γ/IL-4调节脑内小胶质细胞表型及相应细胞因子目的:探索能否通过腹腔注射Th1细胞因子(IFN-γ)或Th2细胞因子(IL-4)调控脑内小胶质细胞的表型方法:根据第一部分结果,在M1比例显著升高的早期分别给予IFN-γ和IL-4以达到进一步促进M1比例升高和抑制M1比例升高的作用,在M2比例显著升高的中期分别给予IFN-γ和IL-4以抑制M2的产生和进一步促进M2比例升高。选用7-8周龄C57BL/6J小鼠,采用匹鲁卡品腹腔注射诱导惊厥持续状态,随机分成6组:早期生理盐水干预组、早期IFN-γ干预组、早期IL-4干预组、中期生理盐水干预组、中期IFN-γ干预组、中期IL-4干预组。应用FCM检测干预后小胶质细胞的表型变化,进一步应用ELISA检测细胞因子IL-1β、IL-4、IL-10的水平,了解干预后不同小胶质细胞表型是否发生了变化,及表型相关细胞因子水平的变化是否与表型一致。结果:(1)腹腔注射细胞因子能够达到增加外周血及脑组织细胞因子浓度的作用。(2)早期补充IFN-γ不能进一步增加SE后M1的比例。中期补充IL-4也无法进一步增加M2的比例。SE后表型相关细胞因子在SE后第15天达到峰值,而早期IFN-γ组、中期IL-4组脑内IL-1β、IL-4、IL-10的峰值水平与生理盐水干预组无显著性差异。(3)早期给予IL-4可以显著抑制SE后早期M1的持续升高。同样的,中期给予IFN-γ抑制了M2的升高。在SE后第15天,早期IL-4组及中期IFN-γ组脑内IL-1β、IL-4、IL-10的峰值水平较生理盐水干预组明显降低。(4)抑制表型发生偏移并不是通过抑制小胶质细胞活化实现的。结论:进一步增加SE后小胶质细胞表型偏移程度的干预措施不能到达预期效果,而抑制SE后小胶质细胞表型偏移的干预措施均成功抑制了表型的偏移。第三部分抑制SE后小胶质细胞表型偏移对癫痫发生及认知功能损伤的作用目的:探索在SE后通过抑制小胶质细胞的表型的偏移能否影响癫痫的发生及脑认知功能损伤。方法:在SE后第50天对实验组(生理盐水干预组、早期IL-4干预组、中期IFN-γ干预组)及对照组(正常对照组、IFN-γ对照组、IL-4对照组)的小鼠植入颅内电极,利用脑电连续检测14天,观察自发性癫痫发作(Spontaneous recurrent seizures,SRS)情况。应用Morris水迷宫检测干预后小鼠的空间学习记忆能力来反应认知功能的变化。结果:在脑电检测过程中,对照组(正常对照组、IFN-γ对照组、IL-4对照组)未出现惊厥发作。早期IL-4干预组、中期IFN-γ干预组的SRS发生率虽然无统计学差异,但是SRS的持续时间、发作频率、严重程度均较生理盐水干预组有所改善。早期IL-4干预组、中期IFN-γ干预组的Morris水迷宫表现也优于生理盐水干预组,但仍不及正常对照组。结论:根据小胶质细胞表型在SE后的变化趋势,精准化的抑制表型的偏移,能够改善SE的预后,降低SRS的持续时间、发作频率及严重程度,并且减轻SE后认知功能损伤。
[Abstract]:The first part is the dynamic change of microglia phenotype in the process of epilepsy: To explore the dynamic changes of microglia phenotypes and phenotypic cytokines in the brain of mice during the different periods of epilepsy. Methods: 7-8 weeks old C57BL/6J mice were selected to be divided into normal control group and experimental group. Status epilepticus (SE) was induced by intraperitoneal injection, and the model was verified by electroencephalogram monitoring. FCM was used to monitor the changes of microglia phenotype in different periods after SE; enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay, ELISA) was used to monitor the differentiation of microglia in different phenotypes after SE. The changes of interleukin -1 beta (Interleukin-1 beta, IL-1 beta), interleukin -4 (Interleukin-4, IL-4), and interleukins -10 (Interleukin-10, IL-10). Results: (1) pilocarpine can induce convulsion in C57BL/6J mice, and the changes of EEG in SE after the onset of epilepsy are in accordance with the changes of electroencephalogram during the various periods of epilepsy. (2) small The phenotypic proportion of microglia in the rat brain changed dynamically in the process of epilepsy after SE. The proportion of M1 microglia increased rapidly after SE, and maintained at a high level until after SE 20 days, and then declined gradually and then dropped to the control level. The proportion of M2 microglia decreased rapidly after SE, and then gradually increased, after SE. The peak was reached about twentieth days and was close to the control level at twenty-eighth days after SE. (3) the trend of the fluctuation trend of the typical cytokine IL-1 beta secreted by M1 microglia in the brain was basically consistent with the change trend of the M1 microglia ratio; the level of the canonical cytokine secreted by M2 microglia, the level of IL-10 and the M2 microglia, were secreted by M2 microglia cells. The change trend of cell proportion is basically consistent. Conclusion: the proportion of different phenotypes of small glial cells in the process of epilepsy after SE changes, the level of phenotypic related cytokines is basically consistent with phenotypic changes, indicating that the phenotypic change of microglia participates in the process of epileptic onset. The second part is intraperitoneally injected with IFN- gamma /IL-4 Microglia phenotypes and corresponding cytokines in the brain: explore whether Th1 cytokines (IFN- gamma) or Th2 cytokine (IL-4) are injected into the brain to regulate the phenotypic method of microglia in the brain: according to the first part, IFN- gamma and IL-4 are given to increase the ratio of M1 to increase the proportion of M1 in the early stage of the significant increase in M1 ratio. The effect of increasing the proportion of M1 was given to IFN- gamma and IL-4 to inhibit the production of M2 and to further promote the increase of M2 ratio in the middle of the M2 ratio. The 7-8 week old C57BL/6J mice were selected to induce the persistent state of convulsion by intraperitoneal injection of pilocarpine, which were randomly divided into 6 groups: early physiological saline intervention group, early IFN- gamma intervention group, early IFN- gamma intervention group, and early IFN- gamma intervention group. Phase IL-4 intervention group, medium-term saline intervention group, medium-term IFN- gamma intervention group and medium-term IL-4 intervention group. The phenotypic changes of microglia were detected by FCM, and the levels of IL-1 beta, IL-4, IL-10 were detected by ELISA, and the changes of the microglia phenotypes after intervention and the phenotype related cytokines were found. The results were as follows: (1) intraperitoneal injection of cytokines could increase the concentration of cytokines in peripheral blood and brain tissue. (2) early supplementation of IFN- gamma could not further increase the proportion of M1 after SE. Medium term supplementation of IL-4 could not further increase the phenotype related cytokines after.SE in fifteenth Tianda after SE. In the early IFN- gamma group, the peak level of IL-1 beta, IL-4, IL-10 in the middle IL-4 group was not significantly different from that in the saline intervention group. (3) the early administration of IL-4 could significantly inhibit the continuous increase of M1 in the early stage of SE. The peak level of IL-4, IL-10 was significantly lower than that in the saline intervention group. (4) the inhibition of phenotypic migration was not achieved by inhibiting the activation of microglia. Conclusion: intervention measures to further increase the phenotypic migration of microglia after SE could not reach the expected effect, and the intervention measures to inhibit the phenotypic migration of microglia after SE were inhibited. The third part inhibits the effect of phenotypic migration of microglia after SE on epilepsy and cognitive impairment: To explore whether the phenotype shift of microglia can affect the occurrence of epilepsy and brain cognitive impairment after SE. Methods: the experimental group (physiological saline dry) at the end of the fiftieth day of SE Pre group, early IL-4 intervention group, medium IFN- gamma intervention group and control group (normal control group, IFN- gamma control group, IL-4 control group) were implanted with intracranial electrodes in mice, and the spontaneous epileptic seizures (Spontaneous recurrent seizures, SRS) were observed for 14 days by electroencephalography. The spatial learning and memory of the mice after the intervention were detected by the Morris water maze. Results: in the process of EEG detection, the control group (normal control group, IFN- gamma control group, IL-4 control group) did not appear convulsive seizures. The incidence of SRS in the early IL-4 intervention group and the mid-term IFN- gamma intervention group was not statistically different, but the duration of SRS, the frequency of attack, and the severity were more than that of the saline dry. In the early IL-4 intervention group, the Morris water maze in the mid-term IFN- gamma intervention group was also better than the normal saline intervention group, but it was still inferior to the normal control group. Conclusion: according to the change trend of the microglia phenotype after SE, the precision of the inhibitory phenotypic migration can improve the prognosis of SE, reduce the duration of SRS, the frequency of the attack and the frequency of the attack. Severity, and reduce cognitive impairment after SE.
【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1

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