發(fā)布時間：2018-06-09 00:01

  本文選題：蛛網(wǎng)膜下腔出血 + 載脂蛋白E��； 參考：《西南醫(yī)科大學》2017年碩士論文

【摘要】：目的:明確載脂蛋白E(Apolipoprotein E,APOE表示基因,apoE表示蛋白)與蛛網(wǎng)膜下腔出血(Subarachnoid hemorrhage,SAH)后早期腦損傷(Early brain injury,EBI)中血腦屏障破壞的相關性,并探討其可能的作用機制。在此基礎上,驗證基于apoE的治療策略(apoE擬肽)是否可以改善SAH后早期的血腦屏障破壞,減輕EBI。旨在進一步揭示SAH后EBI的發(fā)生機制,為尋找治療SAH的新靶點、開發(fā)新的治療藥物提供實驗數(shù)據(jù)和理論依據(jù)支持。方法:1、探討apoE與SAH后EBI中血腦屏障破壞的相關性。125只成年健康野生型雄性C57BL6/J小鼠(20±1.5g;8-10周),隨機分為假手術組(SHAM)和SAH組,SAH組再細分為6小時(WT-6H)、24小時(WT-24H)、48小時(WT-48H)和72小時(WT-72H)4個亞組(n=25)。運用國際公認的血管內(nèi)穿刺法構建野生型C57BL6/J小鼠的SAH模型;運用蛋白質免疫印跡和免疫熒光染色等技術檢測各組小鼠SAH后apo E的表達情況;運用腦含水量檢測、血腦屏障通透性檢測、酶聯(lián)免疫吸附試驗、神經(jīng)功能學評分、疲勞轉棒實驗和磁共振T2加權成像等技術分析apoE與SAH后EBI中血腦屏障破壞的相關性。2、探討apoE減輕SAH后EBI中血腦屏障破壞的機制。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,分別隨機分成野生型假手術組(WT-SHAM)、野生型SAH-48小時組(wt-48h)、敲除鼠假手術組(ko-sham)和敲除鼠sah-48小時組(ko-48h),(n=25)。運用國際公認的血管內(nèi)穿刺法構建小鼠的sah模型;運用蛋白質免疫印跡和免疫熒光染色等技術檢測各組小鼠sah后apoe的表達情況;運用腦含水量檢測、血腦屏障通透性檢測、酶聯(lián)免疫吸附試驗、神經(jīng)功能學評分、疲勞轉棒實驗和磁共振t2加權成像等技術評估apoe基因敲除鼠與野生型小鼠sah后ebi中血腦屏障破壞的差異情況,并探討可能機制。3、探討基于apoe的治療策略是否可以改善sah后ebi中的血腦屏障破壞。105只野生型c57bl6/j小鼠(19.8±1.7g;8-10周)隨機分為假手術組(sham)和sah組,sah組再細分為安慰劑組(wt-w8h)和apoe擬肽cog1410治療組(wc-48h)共2個亞組(n=25)。運用國際公認的血管內(nèi)穿刺法構建野生型c57bl6/j小鼠的sah模型;治療組小鼠予以apoe擬肽cog1410(1mg.kg-1.d-1)尾靜脈注射,安慰劑組予以等量生理鹽水尾靜脈注射。運用蛋白質免疫印跡和免疫熒光染色等技術檢測各組小鼠sah后apoe的表達情況;運用腦含水量檢測、血腦屏障通透性檢測、酶聯(lián)免疫吸附試驗、神經(jīng)功能學評分、疲勞轉棒實驗和磁共振t2加權成像、18f-fdgpet/ct等技術評估apoe擬肽cog1410對sah后早期血腦屏障破壞的改善作用。結果:1、sah后,小鼠腦組織內(nèi)的apoe表達逐漸升高,到出血后48小時達到高峰,72小時開始逐漸下降。運用免疫熒光染色,我們發(fā)現(xiàn)sah后apoe主要由星形膠質細胞表達。進一步研究發(fā)現(xiàn)apoe在sah后毛細血管周圍的表達明顯增加。同時,小鼠的腦水腫、血腦屏障通透性等反映ebi指標也在傷后48小時達到高峰。因此。我們推測apoe對sah后ebi中的血腦屏障破壞具有重要的相關性。2、apoe基因敲除后,小鼠sah后的神經(jīng)功能預后明顯惡化,血腦屏障破壞更加明顯,伊文思藍、免疫球蛋白G(IgG)滲出較野生鼠明顯增加,磁共振T2高信號區(qū)域也較野生鼠明顯增加,差異具有統(tǒng)計學意義(p0.05)。進一步研究發(fā)現(xiàn)apoE可以通過調(diào)控SAH后的EBI中CypA-NF-κB相關的信號通路,從而減輕神經(jīng)炎癥反應,減輕SAH后的血腦屏障破壞。3、給予野生型SAH小鼠apoE擬肽COG1410治療后,小鼠的血腦屏障破壞明顯減少,伊文思藍和免疫球蛋白G(IgG)滲出較安慰劑治療組明顯減少,磁共振T2高信號區(qū)域減少,小鼠神經(jīng)功能明顯改善,差異具有統(tǒng)計學意義(p0.05)。進一步研究發(fā)現(xiàn)apoE擬肽COG1410也可以通過調(diào)控SAH后的EBI中CypA-NF-κB相關的信號通路,從而減輕神經(jīng)炎癥反應,減輕血腦屏障的破壞。結論:apoE可以通過調(diào)控SAH后的EBI中CypA-NF-κB相關的信號通路,減輕神經(jīng)炎癥反應,從而減輕血腦屏障的破壞,對SAH后的EBI產(chǎn)生保護作用。apoE擬肽COG1410在SAH后的EBI中具有和apoE類似的神經(jīng)保護作用,具有較大的臨床轉化潛能,值得進一步研究。
[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.
【學位授予單位】：西南醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.35

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本文編號：1997822