HDAC4磷酸化水平對腦缺血后血管新生的影響及其機(jī)制探討
發(fā)布時(shí)間：2019-06-09 20:52
【摘要】：研究背景與目的： 腦血管疾病、心臟疾病和惡性腫瘤是人類死亡的三大主要疾病。腦卒中發(fā)生后，常導(dǎo)致患者死亡或遺留嚴(yán)重的神經(jīng)功能障礙，給家庭和社會(huì)帶來沉重負(fù)擔(dān)，危害甚大。流行病學(xué)資料數(shù)據(jù)顯示，在全部腦血管疾病中，缺血性腦卒中約占到80%。針對缺血性腦卒中目前仍沒有很好的治療手段，且效果有限。有學(xué)者提出如能盡早促進(jìn)缺血區(qū)新生血管形成恢復(fù)局部血流，不僅有利挽救半影區(qū)的神經(jīng)元,而且為其后功能修復(fù)中的神經(jīng)干細(xì)胞存活、增殖和功能重塑等提供良好的微環(huán)境，最大限度改善預(yù)后。因此如何盡早恢復(fù)血流促進(jìn)新生血管形成對于缺血性卒中的治療具有重大意義。 血管新生（angiogenesis）是指在原有血管基礎(chǔ)上芽生出新的血管、廣泛重建后形成穩(wěn)定血管網(wǎng)絡(luò)的復(fù)雜過程。血管新生受到多種生長因子共同調(diào)控，是多種相關(guān)基因多步驟協(xié)同作用的共同結(jié)果。缺氧誘導(dǎo)因子-l(hypoxia induciblefactor-1, HIF-1)和血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)是研究較早的廣泛應(yīng)用于缺血性疾病研究的調(diào)控因子。HIF-VEGF信號通路是缺血缺氧后調(diào)節(jié)血管新生的重要機(jī)制之一，但其調(diào)控血管新生的關(guān)鍵環(huán)節(jié)還有待進(jìn)一步闡明。 表觀遺傳學(xué)(epigenetics)修飾在血管新生調(diào)控過程中也扮演了非常重要的角色。組蛋白修飾是表觀遺傳修飾中的一種重要的調(diào)節(jié)方式，其中組蛋白乙酰轉(zhuǎn)移酶（histone acetyltransferases，HATs）和組蛋白去乙�；福╤istone deacetylases，HDACs）在組蛋白修飾過程中不可或缺。在組織發(fā)育和腫瘤血管生成研究中發(fā)現(xiàn)，活化的關(guān)鍵HDACs對于血管新生的發(fā)生和完善起到了關(guān)鍵性調(diào)節(jié)作用。HDAC4（histone deacetylase4）是HDACs家族成員之一,存在多個(gè)磷酸化位點(diǎn)。研究發(fā)現(xiàn)HDAC4蛋白磷酸化后能夠從細(xì)胞核轉(zhuǎn)移至細(xì)胞質(zhì)，從而激活下游與血管相關(guān)基因的表達(dá)。然而在發(fā)生缺血性腦卒中時(shí)磷酸化HDAC4是否也參與調(diào)控血管的新生？其調(diào)控機(jī)制如何，其與已知的HIF-VEGF信號通路的又是如何相互影響目前尚不得知。 據(jù)此，本研究通過觀察體內(nèi)外缺血缺氧時(shí)信號通路分子HIF-1α、VEGFa、HDAC4及P-HDAC4632蛋白等變化規(guī)律，初步探討HDAC4磷酸化水平對腦缺血后血管新生過程的可能影響及其機(jī)制，以期深入理解腦卒中后血管新生的分子調(diào)控機(jī)制，并為腦卒中等缺血性疾病的修復(fù)治療提供新的科學(xué)依據(jù)。 研究內(nèi)容和方法： 1.體內(nèi)實(shí)驗(yàn) 1.1根據(jù)缺血再灌注不同時(shí)間點(diǎn)分為24h和48h組。制作大腦中動(dòng)脈栓塞（Middle cerebral artery occlusion，MCAO）模型（參考Longa法），分別于缺血再灌注后24h、48h分批處死各組大鼠，取腦組織標(biāo)本待檢。 1.2腦組織標(biāo)本冰凍切片行免疫組織化學(xué)熒光染色分別檢測24h和48h缺血再灌注后CD31表達(dá)，比較不同時(shí)間點(diǎn)缺血側(cè)及健側(cè)皮質(zhì)微血管密度，以了解缺血區(qū)血管新生情況。 1.3采用Western blot法分別檢測24h和48h缺血再灌注后大腦缺血皮質(zhì)和健側(cè)皮質(zhì)中HIF-1α、VEGFa、HDAC4和P-HDAC4632蛋白表達(dá)水平。2.體外實(shí)驗(yàn) 2.1構(gòu)建HMEC-1細(xì)胞缺氧和藥物干預(yù)模型，隨機(jī)分為常氧組（N）、缺氧組（H）、磷酸化酶抑制劑干預(yù)組（H+G）。 2.2采用Western blot法分別檢測不同組別細(xì)胞內(nèi)HIF-1α、VEGFa、HDAC4和P-HDAC4632蛋白表達(dá)水平。 2.3采用實(shí)時(shí)熒光定量PCR檢測不同組別細(xì)胞內(nèi)VEGF下游成血管相關(guān)基因RCAN2表達(dá)變化。 2.4觀察不同組別HMEC-1細(xì)胞成管能力變化情況。 結(jié)果： 1.體內(nèi)實(shí)驗(yàn) 1.1大鼠腦中動(dòng)脈腦經(jīng)梗塞后均出現(xiàn)程度不等的神經(jīng)功能缺失表現(xiàn)，包括對側(cè)肢體偏癱、眼瞼下垂、行走時(shí)原地轉(zhuǎn)圈和向?qū)?cè)傾斜、提尾向?qū)?cè)側(cè)身等一系列異常反應(yīng)；TTC染色見右側(cè)大腦中動(dòng)脈供血區(qū)域呈蒼白色，而周圍正常腦組織呈紅色；上述表現(xiàn)均提示腦缺血模型制作成功。 1.2免疫組織化學(xué)熒光染色分別檢測24h和48h缺血再灌注后CD31表達(dá)，結(jié)果顯示缺血側(cè)皮質(zhì)微血管密度明顯多于健側(cè)皮質(zhì)，，且缺血再灌注48h后缺血側(cè)半暗區(qū)新生微血管密度顯著高于再灌注24h后。 1.3Western blot檢測結(jié)果表明缺血再灌注24h后，缺血側(cè)半暗區(qū)皮質(zhì)HIF-1α和VEGFa蛋白表達(dá)明顯要高于健側(cè)皮質(zhì)；但是隨著灌注的時(shí)間的延長至48h后，缺血側(cè)半暗區(qū)皮質(zhì)P-HDAC4632蛋白也明顯高于健側(cè)皮質(zhì)；其中各組別總HDAC4蛋白表達(dá)水平無明顯差異性。 2.體外實(shí)驗(yàn) 2.1在體外使用缺氧條件（1%O2濃度、5%CO2濃度）培養(yǎng)HMEC-1（人類微血管內(nèi)皮細(xì)胞），其中一組為單純?nèi)毖踅M（H組），另一組使用藥物G6976預(yù)處理細(xì)胞30min后再缺氧培養(yǎng)（H+G組），均以常氧組（21%O2濃度、5%CO2濃度）為對照組。 2.2缺氧或藥物刺激12h后，各組細(xì)胞在缺氧條件下無論是否添加磷酸化酶抑制劑G6976干預(yù)，Western blot檢測結(jié)果表明HIF-1α和VEGFa蛋白均高表達(dá)。在各組當(dāng)中HDAC4總蛋白表達(dá)水平無明顯差異性，但P-HDAC4632蛋白與HIF-1α和VEGFa蛋白表達(dá)情況并不一致，H組P-HDAC4632明顯高表達(dá)于常氧組，而H+G組P-HDAC4632蛋白表達(dá)減少。 2.3實(shí)時(shí)熒光定量PCR檢測結(jié)果表明，相比常氧對照組，單純?nèi)毖踅MRCAN2基因明顯高表達(dá)；然而使用磷酸化酶抑制劑G6976預(yù)處理之后，RCAN2基因表達(dá)水平明顯減少。 2.4相比較于常氧組，H組HMEC-1細(xì)胞成管能力明顯增強(qiáng)；而H+G組細(xì)胞成管受到限制。 結(jié)論： 本研究的體內(nèi)腦缺血?jiǎng)游锬Ｐ蛯?shí)驗(yàn)結(jié)果顯示，腦缺血后缺血皮質(zhì)區(qū)微血管密度增多，缺血皮質(zhì)區(qū)HIF-1α和VEGFa表達(dá)明顯上調(diào)，同時(shí)伴隨HDAC4蛋白磷酸化水平的明顯變化；進(jìn)一步通過體外血管內(nèi)皮細(xì)胞缺氧實(shí)驗(yàn)證實(shí)，缺氧會(huì)激活血管內(nèi)皮細(xì)胞的HIF-VEGF信號通路，同時(shí)還明顯影響血管內(nèi)皮細(xì)胞的HDAC4磷酸化水平；磷酸化酶抑制劑G6976可顯著抑制HDAC4的磷酸化，并抑制內(nèi)皮細(xì)胞中VEGF下游成血管基因RCAN2的表達(dá)及其成管能力。研究結(jié)果表明，HDAC4蛋白磷酸化水平在腦組織缺血缺氧后血管新生過程中可能發(fā)揮重要調(diào)控作用，其可能是HIF-VEGF信號通路調(diào)控血管新生的重要靶點(diǎn)。
[Abstract]:Background and purpose of the study: Cerebrovascular disease, heart disease and malignant tumor are the three major diseases of human death The occurrence of a stroke often leads to the death of a patient or a serious neurological disorder, which has a heavy burden on the family and society, which is very harmful The data from the epidemiological data show that in all of the cerebrovascular diseases, the ischemic stroke is about 80%. %. There is still no good treatment means for ischemic stroke, and the effect is The present invention provides a good microenvironment for the survival, proliferation and functional remodeling of neural stem cells in the subsequent functional repair. Therefore, how to restore the blood flow as soon as possible to promote the formation of new blood vessels is of great interest to the treatment of ischemic stroke Angiogenesis refers to the formation of a new blood vessel on the basis of the original blood vessel. The blood vessel is co-regulated by a variety of growth factors, and is a co-operation of the multi-step synergistic effect of a plurality of related genes. The results showed that hypoxia-induced factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) were widely used in the study of ischemic diseases. The control factor. HIF-VEGF signaling pathway is one of the important mechanisms for regulating angiogenesis after ischemia and hypoxia, but the key link in the regulation and control of angiogenesis is still to be advanced. The epigenetic modification also plays a very important role in the regulation of angiogenesis. important role. histone modification is an important regulation in epigenetic modification, in which histone acetyltransferase (hats) and histone deacetylases (HDACs) are modified in histone In the study of tissue development and tumor angiogenesis, the key to the activation of HDACs is the key to the occurrence and improvement of angiogenesis. HDAC4 (histone deacetylae4) is one of the members of the HDACs family. Phosphorylation sites. The study found that after the phosphorylation of the HDAC4 protein, it was able to transfer from the nucleus to the cytoplasm, thereby activating the downstream to be associated with the vessel. Expression of the gene. However, the phosphorylation of HDAC4 in the event of an ischemic stroke is also involved in regulation What is the new blood vessel's new regulation mechanism, which interacts with the known HIF-VEGF signaling pathway The changes of the expression of HIF-1, VEGFa, HDAC4 and P-HDAC4632 were studied by observing the changes of the signal pathway molecules HIF-1, VEGFa, HDAC4 and P-HDAC4632 in the body. can influence and mechanism, in order to deeply understand the molecular control mechanism of the blood vessel after stroke, and provide the treatment and treatment of the ischemic diseases such as cerebral apoplexy For the new scientific basis. Study content and method:1. In vivo experiment 1.1 is different according to the ischemia-reperfusion The time points were divided into 24 h and 48 h groups. The expression of CD31 after 24 h and 48 h of ischemia-reperfusion was detected by immunohistochemical and fluorescent staining of the frozen sections in the brain tissue of each group, and the microvessels of the ischemic side and the healthy side were compared at different time points. The density of HIF-1, VEGFa, HDAC4 and P in the cortex of the cerebral ischemia and the cortex of the healthy side were detected by Western blot. -HDA C4632 protein expression level.2. In vitro experiment 2.1, a model of HMEC-1 cell hypoxia and drug intervention was constructed, which was randomly divided into the normal oxygen group (N) and the hypoxia group. (H) The intervention group (H + G) of the phosphorylase inhibitor (H + G). 2.2 The expression of HIF-1, VEGFa and H in different group of cells was detected by Western blot. The expression level of DAC4 and P-HDAC4632 protein. 2.3 The cells of different groups were detected by real-time fluorescence quantitative PCR. The expression of the vascular-related gene RCAN2 in the downstream of the internal VEGF was changed. 2.4 Observation of different groups Results:1. In vivo experiment,1.1.1.1.1. In vivo experiment,1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1. In the middle of the middle cerebral artery, TTC staining showed that the blood supply area of the right cerebral artery was in the sky. The results showed that the microvessel density of the ischemic side was significantly higher than that of the healthy side cortex, and the results showed that the microvessel density of the ischemic side was significantly higher than that of the side cortex. The results of Western blot showed that the expression of HIF-1 and VEGFa protein in the ischemic-side half-dark region was significantly higher than that of the healthy-side cortex after the ischemia-reperfusion for 48 h, but with the time of the perfusion, the expression of the HIF-1 and the VEGFa protein was significantly higher than that of the side-side cortex; however, with the time of the perfusion, the expression of the HIF-1 and the VEGFa protein was significantly higher than that of the side-side cortex. After 48 h, the cortical P-HDAC4632 in the ischemic side The protein is also obviously high. In vitro, there was no significant difference in the level of HDAC4 protein in each group.2. In vitro experiment 2.1, HMEC-1 (human microvascular endothelial cells) were cultured in vitro using an anoxic condition (1% O2 concentration,5% CO2 concentration), one of which was a pure hypoxia group. (Group H), another group treated with drug G6976 for 30 min After hypoxia or drug stimulation for 12 h, the cells of each group were under the condition of hypoxia, whether or not the phosphorylase inhibitor G6 was added after the hypoxia or the drug was stimulated for 12 h. The results of Western blot showed that the expression of HIF-1 and the expression of VEGFa were not significant, but the expression of HDAC4 protein was not consistent with the expression of HIF-1 and VEGFa. P-HDAC4632 is highly expressed in the normal oxygen group, while the expression of P-HDAC4632 protein in the H + G group is decreased. The expression level of the RCAN2 gene was significantly reduced after the pre-treatment with the phosphorylase inhibitor G6976. 2 4. Compared with the normal oxygen group, the tube-forming ability of the HMEC-1 cells in the H group is obviously enhanced; and the cell-forming ability of the H + G group is limited. Conclusion: The experimental results of the in vivo cerebral ischemia animal model of this study show that the microvessel density in the ischemic cortex after cerebral ischemia The expression of HIF-1 and VEGFa in the ischemic cortex is up-regulated, and the expression of HIF-1 and VEGFa in the ischemic cortex is obviously up-regulated, and the HIF-VEGF signal pathway of the vascular endothelial cells can be activated by the hypoxia experiment of the in vitro vascular endothelial cells, and meanwhile, the hypoxia can activate the HIF-VEGF signal path of the vascular endothelial cells. It also significantly affected the level of HDAC4 phosphorylation of vascular endothelial cells; the phosphorylase inhibitor G697 6. The phosphorylation of HDAC4 can be significantly inhibited, and the expression of VEGF downstream of VEGF in the endothelial cells and the expression of the vascular gene RCAN2 and its tube-forming ability are inhibited. The results show that the level of the phosphorylation of HDAC4 protein in the brain tissue
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
本文編號：2495891
[Abstract]:Background and purpose of the study: Cerebrovascular disease, heart disease and malignant tumor are the three major diseases of human death The occurrence of a stroke often leads to the death of a patient or a serious neurological disorder, which has a heavy burden on the family and society, which is very harmful The data from the epidemiological data show that in all of the cerebrovascular diseases, the ischemic stroke is about 80%. %. There is still no good treatment means for ischemic stroke, and the effect is The present invention provides a good microenvironment for the survival, proliferation and functional remodeling of neural stem cells in the subsequent functional repair. Therefore, how to restore the blood flow as soon as possible to promote the formation of new blood vessels is of great interest to the treatment of ischemic stroke Angiogenesis refers to the formation of a new blood vessel on the basis of the original blood vessel. The blood vessel is co-regulated by a variety of growth factors, and is a co-operation of the multi-step synergistic effect of a plurality of related genes. The results showed that hypoxia-induced factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) were widely used in the study of ischemic diseases. The control factor. HIF-VEGF signaling pathway is one of the important mechanisms for regulating angiogenesis after ischemia and hypoxia, but the key link in the regulation and control of angiogenesis is still to be advanced. The epigenetic modification also plays a very important role in the regulation of angiogenesis. important role. histone modification is an important regulation in epigenetic modification, in which histone acetyltransferase (hats) and histone deacetylases (HDACs) are modified in histone In the study of tissue development and tumor angiogenesis, the key to the activation of HDACs is the key to the occurrence and improvement of angiogenesis. HDAC4 (histone deacetylae4) is one of the members of the HDACs family. Phosphorylation sites. The study found that after the phosphorylation of the HDAC4 protein, it was able to transfer from the nucleus to the cytoplasm, thereby activating the downstream to be associated with the vessel. Expression of the gene. However, the phosphorylation of HDAC4 in the event of an ischemic stroke is also involved in regulation What is the new blood vessel's new regulation mechanism, which interacts with the known HIF-VEGF signaling pathway The changes of the expression of HIF-1, VEGFa, HDAC4 and P-HDAC4632 were studied by observing the changes of the signal pathway molecules HIF-1, VEGFa, HDAC4 and P-HDAC4632 in the body. can influence and mechanism, in order to deeply understand the molecular control mechanism of the blood vessel after stroke, and provide the treatment and treatment of the ischemic diseases such as cerebral apoplexy For the new scientific basis. Study content and method:1. In vivo experiment 1.1 is different according to the ischemia-reperfusion The time points were divided into 24 h and 48 h groups. The expression of CD31 after 24 h and 48 h of ischemia-reperfusion was detected by immunohistochemical and fluorescent staining of the frozen sections in the brain tissue of each group, and the microvessels of the ischemic side and the healthy side were compared at different time points. The density of HIF-1, VEGFa, HDAC4 and P in the cortex of the cerebral ischemia and the cortex of the healthy side were detected by Western blot. -HDA C4632 protein expression level.2. In vitro experiment 2.1, a model of HMEC-1 cell hypoxia and drug intervention was constructed, which was randomly divided into the normal oxygen group (N) and the hypoxia group. (H) The intervention group (H + G) of the phosphorylase inhibitor (H + G). 2.2 The expression of HIF-1, VEGFa and H in different group of cells was detected by Western blot. The expression level of DAC4 and P-HDAC4632 protein. 2.3 The cells of different groups were detected by real-time fluorescence quantitative PCR. The expression of the vascular-related gene RCAN2 in the downstream of the internal VEGF was changed. 2.4 Observation of different groups Results:1. In vivo experiment,1.1.1.1.1. In vivo experiment,1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1.1. In the middle of the middle cerebral artery, TTC staining showed that the blood supply area of the right cerebral artery was in the sky. The results showed that the microvessel density of the ischemic side was significantly higher than that of the healthy side cortex, and the results showed that the microvessel density of the ischemic side was significantly higher than that of the side cortex. The results of Western blot showed that the expression of HIF-1 and VEGFa protein in the ischemic-side half-dark region was significantly higher than that of the healthy-side cortex after the ischemia-reperfusion for 48 h, but with the time of the perfusion, the expression of the HIF-1 and the VEGFa protein was significantly higher than that of the side-side cortex; however, with the time of the perfusion, the expression of the HIF-1 and the VEGFa protein was significantly higher than that of the side-side cortex. After 48 h, the cortical P-HDAC4632 in the ischemic side The protein is also obviously high. In vitro, there was no significant difference in the level of HDAC4 protein in each group.2. In vitro experiment 2.1, HMEC-1 (human microvascular endothelial cells) were cultured in vitro using an anoxic condition (1% O2 concentration,5% CO2 concentration), one of which was a pure hypoxia group. (Group H), another group treated with drug G6976 for 30 min After hypoxia or drug stimulation for 12 h, the cells of each group were under the condition of hypoxia, whether or not the phosphorylase inhibitor G6 was added after the hypoxia or the drug was stimulated for 12 h. The results of Western blot showed that the expression of HIF-1 and the expression of VEGFa were not significant, but the expression of HDAC4 protein was not consistent with the expression of HIF-1 and VEGFa. P-HDAC4632 is highly expressed in the normal oxygen group, while the expression of P-HDAC4632 protein in the H + G group is decreased. The expression level of the RCAN2 gene was significantly reduced after the pre-treatment with the phosphorylase inhibitor G6976. 2 4. Compared with the normal oxygen group, the tube-forming ability of the HMEC-1 cells in the H group is obviously enhanced; and the cell-forming ability of the H + G group is limited. Conclusion: The experimental results of the in vivo cerebral ischemia animal model of this study show that the microvessel density in the ischemic cortex after cerebral ischemia The expression of HIF-1 and VEGFa in the ischemic cortex is up-regulated, and the expression of HIF-1 and VEGFa in the ischemic cortex is obviously up-regulated, and the HIF-VEGF signal pathway of the vascular endothelial cells can be activated by the hypoxia experiment of the in vitro vascular endothelial cells, and meanwhile, the hypoxia can activate the HIF-VEGF signal path of the vascular endothelial cells. It also significantly affected the level of HDAC4 phosphorylation of vascular endothelial cells; the phosphorylase inhibitor G697 6. The phosphorylation of HDAC4 can be significantly inhibited, and the expression of VEGF downstream of VEGF in the endothelial cells and the expression of the vascular gene RCAN2 and its tube-forming ability are inhibited. The results show that the level of the phosphorylation of HDAC4 protein in the brain tissue
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.3
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