载脂蛋白E对实验性自身免疫脑脊髓炎血脑屏障通透性的影响

发布时间：2018-09-04 18:18

【摘要】：目的探讨载脂蛋白E（ApoE）对实验性自身免疫脑脊髓炎（EAE）小鼠中的金属蛋白酶9（MMP-9）及其对血脑屏障通透性的影响。方法用髓鞘少突胶质细胞糖蛋白多肽（MOG35-55）为抗原分别诱导C57BL/6J种属的野生型（WT）和ApoE基因敲除型（ApoE-/-）小鼠建立EAE模型，设立E-WT组和E-ApoE-/-组；并分别建立正常对照组（C），C-WT组和C-ApoE-/-组。各组小鼠通过尾静脉注射伊文思蓝（EB）并测定脑组织中EB渗透含量来评估BBB通透性。取脑组织做石蜡切片，，行HE染色观察炎性浸润情况，免疫组化法检测基质金属蛋白酶-9（MMP-9）和基质金属蛋白酶组织抑制剂-1（TIMP-1）的表达。分别应用实时荧光定量RT-PCR、Western blot检测脑组织中MMP-9、TIMP-1和紧密连接蛋白Occludin、Claudin-5的mRNA转录和蛋白表达水平。应用明胶酶谱分析MMP-9活性的表达。结果与E-WT组小鼠比较，E-ApoE-/-组小鼠在EAE发病后平均评分、峰值评分明显升高（P0.05），而潜伏期比较无明显统计意义（P0.05）；E-ApoE-/-组的中枢神经组织炎症细胞侵润明显，同时脑组织中EB的含量也显著高于E-WT组（P0.05）。与正常组小鼠相比，E-ApoE-/-和E-WT组的脑组织中的occludin和claudin-5的mRNA和蛋白表达水平均明显减少（P0.05）；而E-ApoE-/-组的脑组织中的occludin和claudin-5的mRNA和蛋白表达水均低于E-WT组（P0.05）。另外与正常组小鼠相比，E-ApoE-/-组和E-WT组脑组织中的MMP-9的mRNA、蛋白表达水和活性表达均明显升高（P0.05），而E-ApoE-/-组高于E-WT组（P0.05）；与正常组小鼠相比，E-ApoE-/-组和E-WT组脑组织中的TIMP-1的mRNA和蛋白表达水平均降低（P0.05），E-ApoE-/-组表达稍低于E-WT组（P0.05）。结论ApoE的缺乏会恶化EAE的病情进展，并加重血脑屏障完整性的破坏从而促进炎性细胞侵润，而造成血脑屏障完整性破坏的机制可能与增加MMP-9的表达和增强其活性密切相关，说明ApoE在EAE发病过程中对维持血脑屏障完整性起保护作用，这可能成为MS/EAE另一个治疗靶点。
[Abstract]:Objective to investigate the effects of apolipoprotein E (ApoE) on metalloproteinase-9 (MMP-9) and blood-brain barrier permeability in (EAE) mice with experimental autoimmune encephalomyelitis. Methods the wild-type (WT) and ApoE knockout type (ApoE-/-) mice of C57BL/6J species were induced by myelin oligodendrocyte glycoprotein polypeptide (MOG35-55) as antigen to establish EAE model, E-WT group and E-ApoE-r- group, and normal control (C) C-WT group and C-ApoE-r-group respectively. The permeability of BBB was evaluated by intravenous injection of Evans blue (EB) and determination of EB osmotic content in brain tissue. The expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) was detected by immunohistochemical method. The mRNA transcription and protein expression of MMP-9,TIMP-1 and Occludin,Claudin-5 in brain tissue were detected by real-time fluorescence quantitative RT-PCR,Western blot. The expression of MMP-9 activity was analyzed by gelatinase spectrum. Results compared with the E-WT group, the average score and peak score of the E-ApoE-r- group were significantly higher than those of the E-WT group (P0.05), but there was no significant statistical significance in the latency comparison (P0.05). At the same time, the content of EB in brain tissue was significantly higher than that in E-WT group (P0.05). Compared with normal group, the expression of mRNA and protein of occludin and claudin-5 in brain tissue of E-WT group and E-ApoE-r- group were significantly decreased (P0.05), while the mRNA and protein expression of occludin and claudin-5 in E-ApoE-r- group were lower than those in E-WT group (P0.05). In addition, compared with the normal group, the expression of MMP-9 mRNA, protein in the brain tissue of the E-ApoE-r-group and E-WT group was significantly higher than that of the normal group (P0.05), but the expression of mRNA, protein in E-ApoE-r-group was higher than that in the E-WT group (P0.05). Compared with the normal group, the mRNA and protein expression of TIMP-1 in the brain tissue of the E-ApoE-r-group and E-WT group were decreased (P0.05), and the expression of E-ApoE-r- group was slightly lower than that of the E-WT group (P0.05). Conclusion the deficiency of ApoE can aggravate the progression of EAE and aggravate the damage of the integrity of blood-brain barrier, thus promoting the infiltration of inflammatory cells. The mechanism of the damage of blood-brain barrier integrity may be closely related to the increase of MMP-9 expression and the enhancement of its activity. It is suggested that ApoE plays a protective role in maintaining the integrity of blood-brain barrier during the pathogenesis of EAE, which may be another therapeutic target for MS/EAE.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R744.3

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