载脂蛋白E及其拟肽对蛛网膜下腔出血后早期血脑屏障破坏的影响及机制

发布时间：2018-06-09 00:01

本文选题：蛛网膜下腔出血 + 载脂蛋白E　；参考：《西南医科大学》2017年硕士论文

【摘要】：目的:明确载脂蛋白E(Apolipoprotein E,APOE表示基因,apoE表示蛋白)与蛛网膜下腔出血(Subarachnoid hemorrhage,SAH)后早期脑损伤(Early brain injury,EBI)中血脑屏障破坏的相关性,并探讨其可能的作用机制。在此基础上,验证基于apoE的治疗策略(apoE拟肽)是否可以改善SAH后早期的血脑屏障破坏,减轻EBI。旨在进一步揭示SAH后EBI的发生机制,为寻找治疗SAH的新靶点、开发新的治疗药物提供实验数据和理论依据支持。方法:1、探讨apoE与SAH后EBI中血脑屏障破坏的相关性。125只成年健康野生型雄性C57BL6/J小鼠(20±1.5g;8-10周),随机分为假手术组(SHAM)和SAH组,SAH组再细分为6小时(WT-6H)、24小时(WT-24H)、48小时(WT-48H)和72小时(WT-72H)4个亚组(n=25)。运用国际公认的血管内穿刺法构建野生型C57BL6/J小鼠的SAH模型;运用蛋白质免疫印迹和免疫荧光染色等技术检测各组小鼠SAH后apo E的表达情况;运用脑含水量检测、血脑屏障通透性检测、酶联免疫吸附试验、神经功能学评分、疲劳转棒实验和磁共振T2加权成像等技术分析apoE与SAH后EBI中血脑屏障破坏的相关性。2、探讨apoE减轻SAH后EBI中血脑屏障破坏的机制。野生型C57BL6/J小鼠(19.6±1.4g;8-10周)和apoE基因敲除(APOE-/-,KO)小鼠(19.8±1.8g;8-10周)各50只,分别随机分成野生型假手术组(WT-SHAM)、野生型SAH-48小时组(wt-48h)、敲除鼠假手术组(ko-sham)和敲除鼠sah-48小时组(ko-48h),(n=25)。运用国际公认的血管内穿刺法构建小鼠的sah模型;运用蛋白质免疫印迹和免疫荧光染色等技术检测各组小鼠sah后apoe的表达情况;运用脑含水量检测、血脑屏障通透性检测、酶联免疫吸附试验、神经功能学评分、疲劳转棒实验和磁共振t2加权成像等技术评估apoe基因敲除鼠与野生型小鼠sah后ebi中血脑屏障破坏的差异情况,并探讨可能机制。3、探讨基于apoe的治疗策略是否可以改善sah后ebi中的血脑屏障破坏。105只野生型c57bl6/j小鼠(19.8±1.7g;8-10周)随机分为假手术组(sham)和sah组,sah组再细分为安慰剂组(wt-w8h)和apoe拟肽cog1410治疗组(wc-48h)共2个亚组(n=25)。运用国际公认的血管内穿刺法构建野生型c57bl6/j小鼠的sah模型;治疗组小鼠予以apoe拟肽cog1410(1mg.kg-1.d-1)尾静脉注射,安慰剂组予以等量生理盐水尾静脉注射。运用蛋白质免疫印迹和免疫荧光染色等技术检测各组小鼠sah后apoe的表达情况;运用脑含水量检测、血脑屏障通透性检测、酶联免疫吸附试验、神经功能学评分、疲劳转棒实验和磁共振t2加权成像、18f-fdgpet/ct等技术评估apoe拟肽cog1410对sah后早期血脑屏障破坏的改善作用。结果:1、sah后,小鼠脑组织内的apoe表达逐渐升高,到出血后48小时达到高峰,72小时开始逐渐下降。运用免疫荧光染色,我们发现sah后apoe主要由星形胶质细胞表达。进一步研究发现apoe在sah后毛细血管周围的表达明显增加。同时,小鼠的脑水肿、血脑屏障通透性等反映ebi指标也在伤后48小时达到高峰。因此。我们推测apoe对sah后ebi中的血脑屏障破坏具有重要的相关性。2、apoe基因敲除后,小鼠sah后的神经功能预后明显恶化,血脑屏障破坏更加明显,伊文思蓝、免疫球蛋白G(IgG)渗出较野生鼠明显增加,磁共振T2高信号区域也较野生鼠明显增加,差异具有统计学意义(p0.05)。进一步研究发现apoE可以通过调控SAH后的EBI中CypA-NF-κB相关的信号通路,从而减轻神经炎症反应,减轻SAH后的血脑屏障破坏。3、给予野生型SAH小鼠apoE拟肽COG1410治疗后,小鼠的血脑屏障破坏明显减少,伊文思蓝和免疫球蛋白G(IgG)渗出较安慰剂治疗组明显减少,磁共振T2高信号区域减少,小鼠神经功能明显改善,差异具有统计学意义(p0.05)。进一步研究发现apoE拟肽COG1410也可以通过调控SAH后的EBI中CypA-NF-κB相关的信号通路,从而减轻神经炎症反应,减轻血脑屏障的破坏。结论:apoE可以通过调控SAH后的EBI中CypA-NF-κB相关的信号通路,减轻神经炎症反应,从而减轻血脑屏障的破坏,对SAH后的EBI产生保护作用。apoE拟肽COG1410在SAH后的EBI中具有和apoE类似的神经保护作用,具有较大的临床转化潜能,值得进一步研究。
[Abstract]:Objective: to clarify the correlation of the blood brain barrier damage in early brain injury (Early brain injury, EBI) after the apo E (Apolipoprotein E, APOE expression gene, apoE expression protein) and subarachnoid hemorrhage (SAH), and to explore the possible mechanism. Whether or not it can improve the early blood brain barrier damage after SAH, reduce EBI. to further reveal the mechanism of EBI after SAH, and provide experimental data and theoretical support for the search for new targets for the treatment of SAH and the development of new therapeutic drugs. Method: 1, the study of the correlation between apoE and the blood brain barrier damage in EBI in EBI,.125 adult healthy wild type male The sex C57BL6/J mice (20 + 1.5g; 8-10 weeks) were randomly divided into sham operation group (SHAM) and SAH group. The SAH group was subdivided into 6 hours (WT-6H), 24 hours (WT-24H), 48 hours (WT-48H) and 72 hours (WT-72H) 4 subgroups (n=25). The SAH model of wild type C57BL6/J mice was constructed by internationally recognized intravascular puncture; protein immunoblotting and immunofluorescence were used. The expression of apo E after SAH was detected by light staining and other techniques. The correlation of brain water content detection, blood brain barrier permeability test, enzyme linked immunosorbent assay, neurologic function score, fatigue bar test and magnetic resonance T2 weighted imaging were used to analyze the correlation.2 of the blood brain barrier damage in EBI after apoE and SAH, and to explore apoE reduced SAH EBI. The mechanism of the destruction of the middle blood brain barrier. The wild type C57BL6/J mice (19.6 + 1.4g; 8-10 weeks) and apoE gene knockout (APOE-/-, KO) mice (19.8 + 1.8g; 8-10 weeks) each were randomly divided into the wild type sham operation group (WT-SHAM), the wild type SAH-48 hour group (wt-48h), the knockout rat sham operation group (ko-sham) and the sah-48 hour group (ko-48h). The SAH model of mice was constructed with internationally recognized intravascular puncture, and the expression of apoE after SAH was detected by immunoblotting and immunofluorescence staining, and the brain water content detection, blood brain barrier permeability test, enzyme linked immunosorbent assay, deity function score, fatigue stick experiment and magnetic resonance T2 added. Weight imaging and other techniques were used to assess the difference of blood brain barrier damage in ApoE knockout mice and wild type mice after SAH EBI, and to explore the possible mechanism.3, and to explore whether the treatment strategy based on apoE could improve the blood brain barrier in EBI after SAH to destroy.105 only wild type c57bl6/j mice (19.8 + 1.7g; 8-10 weeks) and randomly divided into sham operation group (sham) and SAH. Group SAH was subdivided into 2 subgroups (n=25) of placebo group (wt-w8h) and apoE pseudo peptide cog1410 treatment group (wc-48h). The SAH model of wild type c57bl6/j mice was constructed by internationally recognized intravascular puncture; the mice in the treatment group were injected with APOE pseudo peptide cog1410 (1mg.kg-1.d-1), and the placebo group was given the same amount of saline tail vein injection. The expression of apoE after SAH was detected by using protein immunoblotting and immunofluorescence staining and other techniques, such as brain water content detection, blood brain barrier permeability test, enzyme linked immunosorbent assay, neurologic function score, fatigue stick experiment and magnetic resonance T2 weighted imaging, and 18f-fdgpet/ct techniques to evaluate the APOE peptide cog1410 to SAH Results: after 1, SAH, the expression of apoE in the brain tissue of mice increased gradually, reached the peak at 48 hours after hemorrhage, and gradually decreased after 72 hours. Using immunofluorescence staining, we found that apoE was mainly expressed by astrocytes after SAH. Further study found that apoE was around the capillary after SAH. The expression of brain edema and blood brain barrier permeability in mice reached a peak at 48 hours after injury. Therefore, we speculate that apoE has an important correlation with the destruction of blood brain barrier in EBI after SAH. After the knockout of ApoE gene, the prognosis of the neural work after SAH in mice is obviously worse and the damage of blood brain barrier is more obvious. Evans blue and immunoglobulin G (IgG) exudation increased significantly compared with wild mice, and the high signal region of magnetic resonance T2 was also significantly higher than that of wild mice. The difference was statistically significant (P0.05). Further studies found that apoE could reduce the neuroinflammatory response and reduce the blood brain after SAH by regulating the CypA-NF- kappa B related signaling pathway in EBI after SAH. After the barrier destroyed.3, the blood brain barrier destruction in the wild type SAH mice was significantly reduced after apoE quasi peptide COG1410 treatment. The exudation of Evans blue and immunoglobulin G (IgG) was significantly reduced, the high signal region of magnetic resonance T2 decreased and the neural function of mice improved obviously. The difference was statistically significant (P0.05). Further study of hair was found. The present apoE peptide COG1410 can also regulate the signal pathway related to CypA-NF- kappa B in EBI after SAH, thus alleviating the neuroinflammatory response and reducing the destruction of the blood brain barrier. Conclusion: apoE can reduce the reaction of neuroinflammation by regulating the signal pathway of CypA-NF- kappa B related to SAH after SAH, thus reducing the damage of the blood brain barrier and after SAH. The protective effect of.ApoE peptide COG1410 has a neuroprotective effect similar to apoE in EBI after SAH, and has great potential for clinical transformation. It is worth further study.
【学位授予单位】：西南医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.35

【参考文献】