亚甲蓝对EAE小鼠的治疗作用及机制研究

发布时间：2018-03-08 00:24

本文选题：多发性硬化　切入点：实验性自身免疫性脑脊髓炎　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:研究亚甲蓝(methylene blue,MB)对由髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG)诱导的实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠发病的影响,观察小鼠脾组织中Th17、Treg细胞的比例变化,初步探讨MB对EAE小鼠的治疗作用以及可能免疫机制。方法:1将24只C57BL/6小鼠随机分为EAE组、MB组和control组,每组分别8只。以MOG35-55为抗原免疫,辅以百日咳毒素、完全弗氏佐剂及结核菌素,建立小鼠EAE模型,control组不免疫。免疫当天记为第0天,自免疫第1天开始给药,MB组小鼠给予MB 20mg/kg每日腹腔注射;control组及EAE组小鼠每日给予等体积生理盐水腹腔注射。观察小鼠的发病时间和每日神经功能评分。2将18只C57BL/6小鼠随机分为EAE组、MB组和control组,每组分别6只。以MOG35-55为抗原免疫,辅以百日咳毒素、完全弗氏佐剂及结核菌素,建立小鼠EAE模型,control组不免疫。免疫当天记为第0天,自免疫第1天开始给药,MB组小鼠给予MB 20mg/kg每日腹腔注射;control组及EAE组小鼠每日给予等体积生理盐水腹腔注射。免疫后25天(发病高峰期)将小鼠处死,无菌条件下取出小鼠的脾脏。流式细胞学方法测定小鼠脾组织细胞中Th17和Treg细胞的比例。结果:1各组小鼠发病情况:EAE组和MB组小鼠发病率均为100%,发病高峰期在免疫后25天左右。与EAE组相比,MB组发病高峰期的神经功能评分明显降低(P0.01);MB组的发病时间较EAE组晚(P0.05)。control组小鼠不发病;2各组小鼠脾组织中Treg细胞的比例变化:在发病高峰期,各组小鼠脾组织中Treg细胞比例均有明显差别。与control组相比,EAE组小鼠脾组织中Treg细胞比例明显降低(P0.01);与EAE组和control组相比,MB组小鼠脾组织中Treg细胞比例明显升高(P0.01);3各组小鼠脾组织中Th17细胞的比例变化:在发病高峰期,与control组相比,EAE小鼠脾组织中Th17细胞比例明显升高(P0.05);与EAE组对比,MB组小鼠脾组织中Th17细胞比例明显降低(P0.05);但MB组与control组相比,小鼠脾组织中Th17细胞比例无明显差异(P0.05)。结论:1 MB干预可以降低EAE小鼠的神经功能缺损程度,使EAE小鼠的发病时间推迟。2 MB干预可能通过影响小鼠脾组织内Th17/Treg细胞比例从而使EAE小鼠神经缺损程度减轻,推迟发病时间,但其具体的机制仍待进一步研究。
[Abstract]:Aim: to study the effect of methylene blue (MBB) on the pathogenesis of experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG) in mice, and to observe the percentage of Th17T Treg cells in spleen tissue of mice. Methods 24 C57BL / 6 mice were randomly divided into EAE group (MB group) and control group (8 mice in each group). The mice were immunized with MOG35-55 as antigen and with pertussis toxin. Complete Freund's adjuvant and tuberculin were used to establish EAE model in mice. The control group was not immunized. The day of immunization was recorded as day 0. Since the first day of immunization, mice in the MB-treated group were given intraperitoneal injection of MB 20mg / kg daily intraperitoneal injection of normal saline in the control group and the EAE group. The onset time and daily neurological function score of 18 C57BL / 6 mice were observed. Mice were randomly divided into EAE group and control group. Six mice in each group were immunized with MOG35-55 as antigen, supplemented with pertussis toxin, complete Freund's adjuvant and tuberculin. Since the first day of immunization, mice in MB-treated group were given intraperitoneal injection of MB 20mg / kg daily intraperitoneal injection of control group and EAE group, and mice in EAE group were given intraperitoneal injection of the same volume of normal saline daily. The mice were killed 25 days after immunization (peak period of onset). The percentage of Th17 and Treg cells in spleen tissue cells of mice was determined by flow cytometry. Results the incidence of Th17 and Treg cells in mice in group 1 was 100, and the peak period of disease was in immunity. Compared with the EAE group, the neurological function score of the peak period of the disease in the MB group was significantly lower than that in the EAE group. The percentage of Treg cells in the spleen tissue of the control group was significantly lower than that of the EAE group, and the percentage of Treg cells in the spleen tissue of the mice in the control group was lower than that in the control group. Compared with the control group, the percentage of Treg cells in the spleen tissue of the control group was significantly lower than that of the EAE group and the control group, and the percentage of Treg cells in the spleen tissue of the MB-MB group was significantly higher than that of the EAE group and the control group. The percentage change of Th17 cells in spleen tissue of mice in group A: during the peak period of onset, Compared with control group, the proportion of Th17 cells in spleen tissue of control mice was significantly higher than that of EAE group, and the percentage of Th17 cells in spleen tissue of EAE group was significantly lower than that of EAE group, but the ratio of Th17 cells in MB group was significantly lower than that in control group. There was no significant difference in the proportion of Th17 cells in spleen tissue of mice (P 0.05). Conclusion the intervention of 1: 1 MB can reduce the degree of nerve function defect in EAE mice. Delaying the onset time of EAE mice with the intervention of 2.2MB may reduce the degree of nerve defect and delay the onset time of EAE mice by affecting the proportion of Th17/Treg cells in the spleen tissue of EAE mice, but the specific mechanism remains to be further studied.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R744.51

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