Semaphorin 3A對TBI后繼發(fā)性血腦屏障破壞的作用研究
發(fā)布時間：2018-09-03 14:13
【摘要】：研究目的:創(chuàng)傷性腦損傷(TBI)是神經(jīng)外科領(lǐng)域中死亡率及致殘率都極高的一類疾病,TBI后的繼發(fā)性腦損傷、如顱內(nèi)炎癥反應(yīng)、凝血功能障礙等都對神經(jīng)功能的缺失以至患者的死亡具有促進(jìn)作用,顯著增加原發(fā)性損傷造成的不良后果。這其中TBI后激發(fā)的血腦屏障破壞以及腦水腫的發(fā)生在創(chuàng)傷性腦損傷的致死因素中占有很大比重,其所引起的顱內(nèi)炎癥反應(yīng),顱內(nèi)高壓等病理損害極大地加深TBI所致的神經(jīng)功能障礙。而Semaphorin 3A(SEMA 3A)是SEMAs家族成員中最先被研究的神經(jīng)導(dǎo)向因子,因該蛋白在多種疾病中發(fā)揮重要作用而受到廣泛關(guān)注。SEMA 3A不僅為神經(jīng)軸突生長的負(fù)性調(diào)控蛋白,還在多種病理生理過程中發(fā)揮了抑制血管生成、細(xì)胞遷移與凋亡、腫瘤生長以及調(diào)節(jié)免疫應(yīng)答等作用。更有研究證明SEMA 3A可導(dǎo)致VEGF介導(dǎo)的及非VEGF介導(dǎo)的血管通透性升高作用顯著增加。本實(shí)驗(yàn)利用CCI制作腦創(chuàng)傷模型,探討腦組織中SEMA 3A含量在顱腦損傷(TBI)周期中的變化規(guī)律以及其作用位點(diǎn),通過側(cè)腦室注射SEMA 3A蛋白,觀察分析其對于創(chuàng)傷性腦損傷(TBI)后血腦屏障開放、腦組織含水量的影響。探索SEMA 3A是否為TBI后繼發(fā)性血腦屏障破壞、腦水腫發(fā)生的影響因素。研究方法:1、取C57/BL6小鼠隨機(jī)分為假手術(shù)組、TBI后第1天組、第3天組、第7天組和第14天組,每組各6只,TBI組采用CCI打擊法制作TBI模型,假手術(shù)組只磨取骨窗,不進(jìn)行CCI打擊。Western blot免疫印跡法檢測各組實(shí)驗(yàn)動物腦組織中SEMA 3A含量,免疫熒光法檢測SEMA 3A含量及分布位置。2、將18只C57/BL6小鼠隨機(jī)分為TBI+PBS組、TBI+SEMA 3A組和TBI+SEMA 3A+抗體組,每組6只。TBI+PBS組使用立體定向微量注射儀于側(cè)腦室注射PBS,之后立即采用CCI打擊法制作TBI模型,TBI+SEMA 3A組使用立體定向微量注射儀于側(cè)腦室注射SEMA 3A,之后立即采用CCI打擊法制作TBI模型,TBI+SEMA 3A+抗體組使用立體定向微量注射儀于側(cè)腦室注射SEMA 3A,之后立即采用CCI打擊法制作TBI模型,24h后使用立體定向微量注射儀于側(cè)腦室注射SEMA 3A抗體。在時間點(diǎn)第1天,第3天,第7天,第14天進(jìn)行Mnss評分。鼠于TBI后第3天水腫高峰期后處死,應(yīng)用干濕重法檢測腦組織水分含量,應(yīng)用Evans Blue滲漏法檢測血腦屏障(BBB)破壞情況。研究結(jié)果:Western blot檢測顯示TBI后SEMA 3A含量高于sham組,且TBI后第1天、第3天、第7天、第14天SEMA 3A含量呈先增高后降低的趨勢,且在TBI后第3天達(dá)到高峰,各時間點(diǎn)差異均有統(tǒng)計學(xué)意義(P0.05)。免疫熒光法檢測顯示SEMA 3A含量的變化趨勢與Western blot檢測的結(jié)果一致,且集中分布在創(chuàng)傷灶周圍。m NSS評分顯示在處理后的第3天和第7天,與PBS組相比,SEMA 3A組評分顯著升高,而抗體拮抗組較SEMA 3A組顯著下降,但未及PBS組水平。干濕重法檢測顯示TBI+SEMA 3A組腦組織中水分含量較TBI+PBS組顯著升高,而TBI+SEMA 3A+抗體組腦組織中水分含量較TBI+SEMA 3A組顯著降低,差異有統(tǒng)計學(xué)意義(P0.05)。Evans Blue滲漏法監(jiān)測顯示TBI+SEMA 3A組腦組織中Evans Blue滲漏較TBI+PBS組顯著升高,而TBI+SEMA 3A+抗體組腦組織中Evans Blue滲漏較TBI+SEMA 3A組顯著降低,差異有統(tǒng)計學(xué)意義(P0.05)。研究結(jié)論:小鼠TBI后腦組織中SEMA 3A的含量增加,且SEMA 3A能增加TBI后小鼠的血腦屏障滲漏,促進(jìn)腦水腫的發(fā)生。
[Abstract]:Objective: Traumatic brain injury (TBI) is a kind of disease with high mortality and disability rate in the field of neurosurgery. Secondary brain injury after TBI, such as intracranial inflammation, coagulation dysfunction and so on, can promote the loss of neurological function and even the death of patients, and significantly increase the adverse consequences of primary injury. The destruction of blood-brain barrier and the occurrence of brain edema after middle TBI play an important role in the fatal factors of traumatic brain injury. The pathological lesions such as intracranial inflammation and intracranial hypertension caused by middle TBI greatly deepen the neurological dysfunction caused by TBI. Scanning electron microscopy (SEMA) 3A is not only a negative regulator of axon growth, but also plays a role in inhibiting angiogenesis, cell migration and apoptosis, tumor growth and regulating immune response in a variety of pathophysiological processes. In this study, we used CCI to make a model of brain trauma to investigate the changes of the content of SEMA 3A in brain tissues during the cycle of brain injury (TBI) and its action sites. The effect of SEMA 3A protein on blood after traumatic brain injury (TBI) was observed and analyzed by intraventricular injection. Methods: 1. C57 / BL6 mice were randomly divided into sham operation group, 1 day after TBI group, 3 day group, 7 day group and 14 day group, each group had 6 mice, TBI group was made TBI model by CCI hit method, sham operation group. Western blot and immunofluorescence were used to detect the content and distribution of SEMA 3A. Eighteen C57/BL6 mice were randomly divided into TBI+PBS group, TBI+SEMA 3A group and TBI+SEMA 3A+antibody group, with 6 mice in each group. PBS was injected into the lateral ventricle with a volume injector, then TBI model was made by CCI percussion method immediately. SEMA 3A was injected into the lateral ventricle with a stereotactic microinjector in the TBI+SEMA 3A group, and then TBI model was made by CCI percussion method immediately. SEMA 3A 3A was injected into the lateral ventricle with a stereotactic microinjector in the TBI+SEMA 3A+antibody group, and then CCA 3A was injected into the lateral ventricle with a stereotactic micro The mice were sacrificed at the 3rd day after TBI. The brain water content was measured by dry and wet weight method. The blood brain barrier (BBB) was detected by Evans Blue leakage method. Results: Western blot showed that the content of SEMA 3A in TBI group was higher than that in sham group, and the content of SEMA 3A increased first and then decreased on the 1st, 3rd, 7th and 14th day after TBI, and reached the peak on the 3rd day after TBI, and the difference was statistically significant (P 0.05). Compared with PBS group, the score of SEMA 3A group was significantly higher, while that of antibody antagonist group was significantly lower than that of SEMA 3A group, but not as high as that of PBS group. The water content in brain tissue of TBI + SEMA 3A + antibody group was significantly lower than that of TBI + SEMA 3A group (P 0.05). Evans Blue leakage monitoring showed that Evans Blue leakage in brain tissue of TBI + SEMA 3A group was significantly higher than that of TBI + PBS group, while Evans Blue leakage in brain tissue of TBI + SEMA 3A + antibody group was significantly higher than that of TBI + SEMA 3A group. Conclusion: The content of SEMA 3A in brain tissue of mice after TBI increased, and SEMA 3A could increase the blood-brain barrier leakage of mice after TBI and promote the occurrence of brain edema.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.15
本文編號：2220206
[Abstract]:Objective: Traumatic brain injury (TBI) is a kind of disease with high mortality and disability rate in the field of neurosurgery. Secondary brain injury after TBI, such as intracranial inflammation, coagulation dysfunction and so on, can promote the loss of neurological function and even the death of patients, and significantly increase the adverse consequences of primary injury. The destruction of blood-brain barrier and the occurrence of brain edema after middle TBI play an important role in the fatal factors of traumatic brain injury. The pathological lesions such as intracranial inflammation and intracranial hypertension caused by middle TBI greatly deepen the neurological dysfunction caused by TBI. Scanning electron microscopy (SEMA) 3A is not only a negative regulator of axon growth, but also plays a role in inhibiting angiogenesis, cell migration and apoptosis, tumor growth and regulating immune response in a variety of pathophysiological processes. In this study, we used CCI to make a model of brain trauma to investigate the changes of the content of SEMA 3A in brain tissues during the cycle of brain injury (TBI) and its action sites. The effect of SEMA 3A protein on blood after traumatic brain injury (TBI) was observed and analyzed by intraventricular injection. Methods: 1. C57 / BL6 mice were randomly divided into sham operation group, 1 day after TBI group, 3 day group, 7 day group and 14 day group, each group had 6 mice, TBI group was made TBI model by CCI hit method, sham operation group. Western blot and immunofluorescence were used to detect the content and distribution of SEMA 3A. Eighteen C57/BL6 mice were randomly divided into TBI+PBS group, TBI+SEMA 3A group and TBI+SEMA 3A+antibody group, with 6 mice in each group. PBS was injected into the lateral ventricle with a volume injector, then TBI model was made by CCI percussion method immediately. SEMA 3A was injected into the lateral ventricle with a stereotactic microinjector in the TBI+SEMA 3A group, and then TBI model was made by CCI percussion method immediately. SEMA 3A 3A was injected into the lateral ventricle with a stereotactic microinjector in the TBI+SEMA 3A+antibody group, and then CCA 3A was injected into the lateral ventricle with a stereotactic micro The mice were sacrificed at the 3rd day after TBI. The brain water content was measured by dry and wet weight method. The blood brain barrier (BBB) was detected by Evans Blue leakage method. Results: Western blot showed that the content of SEMA 3A in TBI group was higher than that in sham group, and the content of SEMA 3A increased first and then decreased on the 1st, 3rd, 7th and 14th day after TBI, and reached the peak on the 3rd day after TBI, and the difference was statistically significant (P 0.05). Compared with PBS group, the score of SEMA 3A group was significantly higher, while that of antibody antagonist group was significantly lower than that of SEMA 3A group, but not as high as that of PBS group. The water content in brain tissue of TBI + SEMA 3A + antibody group was significantly lower than that of TBI + SEMA 3A group (P 0.05). Evans Blue leakage monitoring showed that Evans Blue leakage in brain tissue of TBI + SEMA 3A group was significantly higher than that of TBI + PBS group, while Evans Blue leakage in brain tissue of TBI + SEMA 3A + antibody group was significantly higher than that of TBI + SEMA 3A group. Conclusion: The content of SEMA 3A in brain tissue of mice after TBI increased, and SEMA 3A could increase the blood-brain barrier leakage of mice after TBI and promote the occurrence of brain edema.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R651.15
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，本文編號：2220206
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