血管内皮生长因子对小鼠卒中后血脑屏障的影响及作用机制研究

发布时间：2019-05-18 10:32

【摘要】：背景与目的脑卒中是世界范围内致死和致残的主要疾病之一,其中急性缺血性是最主要的类型,发病率约占脑卒中的60-80%,其高发病率、高复发率、高致残率、高死亡率严重威胁着人类的生活质量。在过去几十年中,神经科学界一直致力于研究脑卒中的治疗方法,也在动物实验中发现了许多令人振奋的有效治疗手段,比如亚低温治疗,缺血预适应,干细胞治疗,神经保护剂,神经营养因子,一氧化氮,经颅激光治疗,血管内皮生长因子(vascular endothelial growth factor,VEGF)等。VEGF具有保护神经细胞、促进神经发生和神经突再生、促进血管生成恢复缺血脑组织血流灌注和帮助缺血性脑损伤后修复的作用,是一种很有前景的治疗缺血性卒中药物。然而,许多基础和临床前试验证明了VEGF治疗缺血性卒中的高风险收益比,并因此阻碍了VEGF进入卒中治疗的临床试验。造成高风险收益比的一个重要原因是其严重的副作用,即加重卒中后血脑屏障的渗漏、脑水肿以及出血转化。血脑屏障是神经血管单元的一个重要元素,它在维持中枢神经系统内环境的稳定中起关键作用。缺血性脑卒中发生后,血脑屏障被破坏,导致许多对神经系统有害的血清蛋白、炎性细胞等进入大脑,进一步加重缺血损伤。预防和阻止VEGF力重血脑屏障破坏和脑水肿有助于降低VEGF治疗卒中的风险收益。为此,我们必须厘清VEGF加重血脑屏障破坏的机制。方法将C57BL/6小鼠分为三组：假手术(Sham)组、大脑中动脉闭塞(middlecerebral artery occlusion, MCAO)模型组和VEGF组,Sham组小鼠进行造模手术,但线栓未堵住大脑中动脉开口处,MCAO组小鼠进行MCAO模型手术,复灌3h后梗塞侧脑室注射PBS, VEGF组造模复灌3h后于梗塞侧给予VEGF。为验证VEGF对缺血性脑卒中后血脑屏障渗透性的影响及对卒中预后的作用,我们测定了卒中24小时后伊文斯蓝(Evans Blue, EB)渗漏量、免疫球蛋白(immunoglobulin, IgG)渗漏量、脑水肿的程度、梗死体积(TTC法)、改良的神经功能缺损评分(nodified Neurological Severity Scores,mNSS)、贴纸去除试验以及加速滚轴试验。为了探索VEGF影响血脑屏障完整性的机制,我们应用包含39430个编码基因的小鼠基因表达谱芯片SurePrint G3 Mouse Gene Expression 8x60K Microarray检测VEGF对卒中后基因表达的影响,试图在这些基因当中找到与血脑屏障完整性相关的基因从而进一步分析机制。两组间信号比值(Fold Change)≥2或0.5,且组间P值0.05的则判定为有显著性差异。之后我们通过实时定量PCR验证了基因芯片的部分结果,并通过生物信息学方法找出可能的调控通路。结果三组间右侧大脑半球(缺血侧)的EB渗漏量、IgG渗漏量和脑组织含水量有显著差异,MCAO组显著高于Sham组,而VEGF组显著高于MCAO组。梗死体积、加速滚轴试验和mNSS评分MCAO组和 VEGF组间无显着差异。芯片结果显示脑缺血显著影响了3381个基因的表达,而VEGF组与MCAO组相比较38个基因表达出现了显著变化,这38个基因中有15个在MCAO组与Sham组的比较中也有改变,它们的功能涉及到细胞分化、调节免疫系统反应、急性期应答等等。在这15个基因中,血清类黏蛋白(orosomucoid, ORM)是与血脑屏障通透性明确相关的基因,也是变化最明显的。本实验应用实时荧光定量PCR、免疫印迹(Western blotting)、免疫组化和免疫荧光证实了ORM1 (ORM最主要成员)表达的变化。生物信息学分析显示ORM1启动子上有2个NF-κB的结合位点,我们应用凝胶超迁移实验证实了NF-κB可以结合于ORM1启动子。凝胶迁移实验结果显示VEGF抑制了NF-κB的DNA结合活性。此外,脑缺血诱导了经典NF-κB途径中的关键因子(IKKα、IKKβ、p-IKKα、IκBα、p-IκBα、p65、p-p65)表达升高,而VEGF抑制了它们的表达。结论VEGF加重了卒中后血脑屏障的破坏,通过抑制NF-κB通路的激活而下调血清类黏蛋白的表达是其中可能的机制之一。
[Abstract]:Background and Objective Stroke is one of the main causes of death and maiming in the world, in which acute ischemic is the most important type, the morbidity is about 60-80% of the stroke, the high incidence rate, the high recurrence rate, the high disability rate and the high death rate seriously threaten the quality of life of the human. In the past few decades, the neuroscientific community has been working to study the treatment of stroke, and many exciting and effective means of treatment, such as sublow-temperature therapy, ischemic preconditioning, stem cell therapy, neuroprotective agents, and neurotrophic factors, have also been found in animal experiments. Nitric oxide, transcranial laser treatment, vascular endothelial growth factor (VEGF), etc. VEGF has the effects of protecting nerve cells, promoting nerve generation and neurite regeneration, promoting blood vessel generation and restoring blood flow perfusion of ischemic brain tissue and helping to repair post-ischemic brain injury, and is a promising medicine for treating ischemic stroke. However, many of the basic and pre-clinical trials have demonstrated the high-risk gain ratio of VEGF in the treatment of ischemic stroke, and thus blocks the clinical trials of VEGF into stroke therapy. An important reason for the high-risk income ratio is its serious side effects, i.e., the leakage of the blood-brain barrier after stroke, the brain edema, and the transformation of the bleeding. The blood-brain barrier is an important element of the neurovascular unit that plays a key role in maintaining the stability of the central nervous system. After the ischemic stroke, the blood-brain barrier is destroyed, leading to a number of serum proteins, inflammatory cells and the like which are harmful to the nervous system to enter the brain, and further aggravate the ischemic injury. Prevention and prevention of VEGF-induced brain-brain barrier destruction and brain edema can help to reduce the risk of VEGF in the treatment of stroke. To this end, we have to clarify the mechanism of VEGF-weighted blood-brain barrier destruction. Methods C57BL/6 mice were divided into three groups: sham operation (Sham) group, middle cerebral artery occlusion (MCAO) model group and VEGF group. After 3 h of re-irrigation, the lateral ventricle was injected with PBS, and VEGF was given to the infarction side after 3 h of model-forming and re-irrigation of the VEGF group. In order to verify the effect of VEGF on the permeability of blood-brain barrier after ischemic stroke and the effect on the prognosis of stroke, we measured the leakage of Evans Blue (EB), the leakage of immunoglobulin (IgG), the degree of cerebral edema and the volume of infarction (TTC) after 24 hours of stroke. Modified Neurological Severity Score (mNSS), sticker removal test, and accelerated roller test. To explore the mechanism of VEGF to affect the integrity of the blood-brain barrier, we used a mouse gene expression profile chip SurePrint G3 Mouse Gene Expression 8x60K Microarray containing 39430 encoded genes to detect the effect of VEGF on post-stroke gene expression, An attempt was made to find a gene related to the integrity of the blood-brain barrier among these genes to further analyze the mechanism. The inter-group signal ratio (Bold Change) was either 2 or 0.5, and there was a significant difference between the two groups of P-values of 0.05. After that, we validated the partial results of the gene chip by real-time quantitative PCR and found the possible regulatory pathways through the bioinformatics method. Results The amount of EB leakage, the amount of IgG leakage and the water content of the brain in the right hemisphere (ischemic side) of the three groups were significantly different, and the group of MCAO was significantly higher than that of the sham group, while the VEGF group was significantly higher than that of the MCAO group. There was no significant difference between the infarct volume, the accelerated roller test, and the mNSS score of the MCAO group and the VEGF group. The results showed that the cerebral ischemia significantly affected the expression of 3381 genes, while the expression of 38 genes in the VEGF group and the MCAO group showed a significant change, among which 15 of the 38 genes were also changed in the comparison of the MCAO group and the Sham group, and their functions involved the differentiation of the cells, Regulating immune system response, acute phase response, etc. In these 15 genes, the serum-like mucin (ORM) is a gene that is clearly related to the permeability of the blood-brain barrier, and is also the most obvious. The changes of the expression of ORM1 (the primary member of ORM) were confirmed by real-time fluorescence quantitative PCR, Western blotting, immunohistochemistry and immunofluorescence. Bioinformatics analysis shows that there are two binding sites of NF-B-B on the ORM1 promoter, and we have confirmed that NF-B can be bound to the ORM1 promoter by gel supermigration experiments. The results of gel migration showed that VEGF inhibited the DNA binding activity of NF-B. In addition, the expression of key factors (IKK, IKK, p-IKK, I, B, p-I, B, p65, p-p65) in the classical NF-B pathway was induced by cerebral ischemia, and the expression of VEGF was inhibited by VEGF. Conclusion VEGF aggravated the destruction of blood-brain barrier after stroke, and the down-regulation of the expression of serum-like mucin was one of the possible mechanisms by inhibiting the activation of NF-B-B pathway.
【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R743.3

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