Semaphorin 3A对TBI后继发性血脑屏障破坏的作用研究

发布时间：2018-09-03 14:13

【摘要】：研究目的:创伤性脑损伤(TBI)是神经外科领域中死亡率及致残率都极高的一类疾病,TBI后的继发性脑损伤、如颅内炎症反应、凝血功能障碍等都对神经功能的缺失以至患者的死亡具有促进作用,显著增加原发性损伤造成的不良后果。这其中TBI后激发的血脑屏障破坏以及脑水肿的发生在创伤性脑损伤的致死因素中占有很大比重,其所引起的颅内炎症反应,颅内高压等病理损害极大地加深TBI所致的神经功能障碍。而Semaphorin 3A(SEMA 3A)是SEMAs家族成员中最先被研究的神经导向因子,因该蛋白在多种疾病中发挥重要作用而受到广泛关注。SEMA 3A不仅为神经轴突生长的负性调控蛋白,还在多种病理生理过程中发挥了抑制血管生成、细胞迁移与凋亡、肿瘤生长以及调节免疫应答等作用。更有研究证明SEMA 3A可导致VEGF介导的及非VEGF介导的血管通透性升高作用显著增加。本实验利用CCI制作脑创伤模型,探讨脑组织中SEMA 3A含量在颅脑损伤(TBI)周期中的变化规律以及其作用位点,通过侧脑室注射SEMA 3A蛋白,观察分析其对于创伤性脑损伤(TBI)后血脑屏障开放、脑组织含水量的影响。探索SEMA 3A是否为TBI后继发性血脑屏障破坏、脑水肿发生的影响因素。研究方法:1、取C57/BL6小鼠随机分为假手术组、TBI后第1天组、第3天组、第7天组和第14天组,每组各6只,TBI组采用CCI打击法制作TBI模型,假手术组只磨取骨窗,不进行CCI打击。Western blot免疫印迹法检测各组实验动物脑组织中SEMA 3A含量,免疫荧光法检测SEMA 3A含量及分布位置。2、将18只C57/BL6小鼠随机分为TBI+PBS组、TBI+SEMA 3A组和TBI+SEMA 3A+抗体组,每组6只。TBI+PBS组使用立体定向微量注射仪于侧脑室注射PBS,之后立即采用CCI打击法制作TBI模型,TBI+SEMA 3A组使用立体定向微量注射仪于侧脑室注射SEMA 3A,之后立即采用CCI打击法制作TBI模型,TBI+SEMA 3A+抗体组使用立体定向微量注射仪于侧脑室注射SEMA 3A,之后立即采用CCI打击法制作TBI模型,24h后使用立体定向微量注射仪于侧脑室注射SEMA 3A抗体。在时间点第1天,第3天,第7天,第14天进行Mnss评分。鼠于TBI后第3天水肿高峰期后处死,应用干湿重法检测脑组织水分含量,应用Evans Blue渗漏法检测血脑屏障(BBB)破坏情况。研究结果:Western blot检测显示TBI后SEMA 3A含量高于sham组,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趋势,且在TBI后第3天达到高峰,各时间点差异均有统计学意义(P0.05)。免疫荧光法检测显示SEMA 3A含量的变化趋势与Western blot检测的结果一致,且集中分布在创伤灶周围。m NSS评分显示在处理后的第3天和第7天,与PBS组相比,SEMA 3A组评分显著升高,而抗体拮抗组较SEMA 3A组显著下降,但未及PBS组水平。干湿重法检测显示TBI+SEMA 3A组脑组织中水分含量较TBI+PBS组显著升高,而TBI+SEMA 3A+抗体组脑组织中水分含量较TBI+SEMA 3A组显著降低,差异有统计学意义(P0.05)。Evans Blue渗漏法监测显示TBI+SEMA 3A组脑组织中Evans Blue渗漏较TBI+PBS组显著升高,而TBI+SEMA 3A+抗体组脑组织中Evans Blue渗漏较TBI+SEMA 3A组显著降低,差异有统计学意义(P0.05)。研究结论:小鼠TBI后脑组织中SEMA 3A的含量增加,且SEMA 3A能增加TBI后小鼠的血脑屏障渗漏,促进脑水肿的发生。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15

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