血管內皮生長因子對小鼠卒中后血腦屏障的影響及作用機制研究
發(fā)布時間：2019-05-18 10:32
【摘要】：背景與目的腦卒中是世界范圍內致死和致殘的主要疾病之一,其中急性缺血性是最主要的類型,發(fā)病率約占腦卒中的60-80%,其高發(fā)病率、高復發(fā)率、高致殘率、高死亡率嚴重威脅著人類的生活質量。在過去幾十年中,神經科學界一直致力于研究腦卒中的治療方法,也在動物實驗中發(fā)現了許多令人振奮的有效治療手段,比如亞低溫治療,缺血預適應,干細胞治療,神經保護劑,神經營養(yǎng)因子,一氧化氮,經顱激光治療,血管內皮生長因子(vascular endothelial growth factor,VEGF)等。VEGF具有保護神經細胞、促進神經發(fā)生和神經突再生、促進血管生成恢復缺血腦組織血流灌注和幫助缺血性腦損傷后修復的作用,是一種很有前景的治療缺血性卒中藥物。然而,許多基礎和臨床前試驗證明了VEGF治療缺血性卒中的高風險收益比,并因此阻礙了VEGF進入卒中治療的臨床試驗。造成高風險收益比的一個重要原因是其嚴重的副作用,即加重卒中后血腦屏障的滲漏、腦水腫以及出血轉化。血腦屏障是神經血管單元的一個重要元素,它在維持中樞神經系統(tǒng)內環(huán)境的穩(wěn)定中起關鍵作用。缺血性腦卒中發(fā)生后,血腦屏障被破壞,導致許多對神經系統(tǒng)有害的血清蛋白、炎性細胞等進入大腦,進一步加重缺血損傷。預防和阻止VEGF力重血腦屏障破壞和腦水腫有助于降低VEGF治療卒中的風險收益。為此,我們必須厘清VEGF加重血腦屏障破壞的機制。方法將C57BL/6小鼠分為三組：假手術(Sham)組、大腦中動脈閉塞(middlecerebral artery occlusion, MCAO)模型組和VEGF組,Sham組小鼠進行造模手術,但線栓未堵住大腦中動脈開口處,MCAO組小鼠進行MCAO模型手術,復灌3h后梗塞側腦室注射PBS, VEGF組造模復灌3h后于梗塞側給予VEGF。為驗證VEGF對缺血性腦卒中后血腦屏障滲透性的影響及對卒中預后的作用,我們測定了卒中24小時后伊文斯藍(Evans Blue, EB)滲漏量、免疫球蛋白(immunoglobulin, IgG)滲漏量、腦水腫的程度、梗死體積(TTC法)、改良的神經功能缺損評分(nodified Neurological Severity Scores,mNSS)、貼紙去除試驗以及加速滾軸試驗。為了探索VEGF影響血腦屏障完整性的機制,我們應用包含39430個編碼基因的小鼠基因表達譜芯片SurePrint G3 Mouse Gene Expression 8x60K Microarray檢測VEGF對卒中后基因表達的影響,試圖在這些基因當中找到與血腦屏障完整性相關的基因從而進一步分析機制。兩組間信號比值(Fold Change)≥2或0.5,且組間P值0.05的則判定為有顯著性差異。之后我們通過實時定量PCR驗證了基因芯片的部分結果,并通過生物信息學方法找出可能的調控通路。結果三組間右側大腦半球(缺血側)的EB滲漏量、IgG滲漏量和腦組織含水量有顯著差異,MCAO組顯著高于Sham組,而VEGF組顯著高于MCAO組。梗死體積、加速滾軸試驗和mNSS評分MCAO組和 VEGF組間無顯著差異。芯片結果顯示腦缺血顯著影響了3381個基因的表達,而VEGF組與MCAO組相比較38個基因表達出現了顯著變化,這38個基因中有15個在MCAO組與Sham組的比較中也有改變,它們的功能涉及到細胞分化、調節(jié)免疫系統(tǒng)反應、急性期應答等等。在這15個基因中,血清類黏蛋白(orosomucoid, ORM)是與血腦屏障通透性明確相關的基因,也是變化最明顯的。本實驗應用實時熒光定量PCR、免疫印跡(Western blotting)、免疫組化和免疫熒光證實了ORM1 (ORM最主要成員)表達的變化。生物信息學分析顯示ORM1啟動子上有2個NF-κB的結合位點,我們應用凝膠超遷移實驗證實了NF-κB可以結合于ORM1啟動子。凝膠遷移實驗結果顯示VEGF抑制了NF-κB的DNA結合活性。此外,腦缺血誘導了經典NF-κB途徑中的關鍵因子(IKKα、IKKβ、p-IKKα、IκBα、p-IκBα、p65、p-p65)表達升高,而VEGF抑制了它們的表達。結論VEGF加重了卒中后血腦屏障的破壞,通過抑制NF-κB通路的激活而下調血清類黏蛋白的表達是其中可能的機制之一。
[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.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743.3
[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.
【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2014
【分類號】：R743.3
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