HDAC4磷酸化水平对脑缺血后血管新生的影响及其机制探讨
发布时间：2019-06-09 20:52
【摘要】：研究背景与目的： 脑血管疾病、心脏疾病和恶性肿瘤是人类死亡的三大主要疾病。脑卒中发生后，常导致患者死亡或遗留严重的神经功能障碍，给家庭和社会带来沉重负担，危害甚大。流行病学资料数据显示，在全部脑血管疾病中，缺血性脑卒中约占到80%。针对缺血性脑卒中目前仍没有很好的治疗手段，且效果有限。有学者提出如能尽早促进缺血区新生血管形成恢复局部血流，不仅有利挽救半影区的神经元,而且为其后功能修复中的神经干细胞存活、增殖和功能重塑等提供良好的微环境，最大限度改善预后。因此如何尽早恢复血流促进新生血管形成对于缺血性卒中的治疗具有重大意义。 血管新生（angiogenesis）是指在原有血管基础上芽生出新的血管、广泛重建后形成稳定血管网络的复杂过程。血管新生受到多种生长因子共同调控，是多种相关基因多步骤协同作用的共同结果。缺氧诱导因子-l(hypoxia induciblefactor-1, HIF-1)和血管内皮生长因子(vascular endothelial growth factor, VEGF)是研究较早的广泛应用于缺血性疾病研究的调控因子。HIF-VEGF信号通路是缺血缺氧后调节血管新生的重要机制之一，但其调控血管新生的关键环节还有待进一步阐明。 表观遗传学(epigenetics)修饰在血管新生调控过程中也扮演了非常重要的角色。组蛋白修饰是表观遗传修饰中的一种重要的调节方式，其中组蛋白乙酰转移酶（histone acetyltransferases，HATs）和组蛋白去乙酰化酶（histone deacetylases，HDACs）在组蛋白修饰过程中不可或缺。在组织发育和肿瘤血管生成研究中发现，活化的关键HDACs对于血管新生的发生和完善起到了关键性调节作用。HDAC4（histone deacetylase4）是HDACs家族成员之一,存在多个磷酸化位点。研究发现HDAC4蛋白磷酸化后能够从细胞核转移至细胞质，从而激活下游与血管相关基因的表达。然而在发生缺血性脑卒中时磷酸化HDAC4是否也参与调控血管的新生？其调控机制如何，其与已知的HIF-VEGF信号通路的又是如何相互影响目前尚不得知。 据此，本研究通过观察体内外缺血缺氧时信号通路分子HIF-1α、VEGFa、HDAC4及P-HDAC4632蛋白等变化规律，初步探讨HDAC4磷酸化水平对脑缺血后血管新生过程的可能影响及其机制，以期深入理解脑卒中后血管新生的分子调控机制，并为脑卒中等缺血性疾病的修复治疗提供新的科学依据。 研究内容和方法： 1.体内实验 1.1根据缺血再灌注不同时间点分为24h和48h组。制作大脑中动脉栓塞（Middle cerebral artery occlusion，MCAO）模型（参考Longa法），分别于缺血再灌注后24h、48h分批处死各组大鼠，取脑组织标本待检。 1.2脑组织标本冰冻切片行免疫组织化学荧光染色分别检测24h和48h缺血再灌注后CD31表达，比较不同时间点缺血侧及健侧皮质微血管密度，以了解缺血区血管新生情况。 1.3采用Western blot法分别检测24h和48h缺血再灌注后大脑缺血皮质和健侧皮质中HIF-1α、VEGFa、HDAC4和P-HDAC4632蛋白表达水平。2.体外实验 2.1构建HMEC-1细胞缺氧和药物干预模型，随机分为常氧组（N）、缺氧组（H）、磷酸化酶抑制剂干预组（H+G）。 2.2采用Western blot法分别检测不同组别细胞内HIF-1α、VEGFa、HDAC4和P-HDAC4632蛋白表达水平。 2.3采用实时荧光定量PCR检测不同组别细胞内VEGF下游成血管相关基因RCAN2表达变化。 2.4观察不同组别HMEC-1细胞成管能力变化情况。 结果： 1.体内实验 1.1大鼠脑中动脉脑经梗塞后均出现程度不等的神经功能缺失表现，包括对侧肢体偏瘫、眼睑下垂、行走时原地转圈和向对侧倾斜、提尾向对侧侧身等一系列异常反应；TTC染色见右侧大脑中动脉供血区域呈苍白色，而周围正常脑组织呈红色；上述表现均提示脑缺血模型制作成功。 1.2免疫组织化学荧光染色分别检测24h和48h缺血再灌注后CD31表达，结果显示缺血侧皮质微血管密度明显多于健侧皮质，，且缺血再灌注48h后缺血侧半暗区新生微血管密度显著高于再灌注24h后。 1.3Western blot检测结果表明缺血再灌注24h后，缺血侧半暗区皮质HIF-1α和VEGFa蛋白表达明显要高于健侧皮质；但是随着灌注的时间的延长至48h后，缺血侧半暗区皮质P-HDAC4632蛋白也明显高于健侧皮质；其中各组别总HDAC4蛋白表达水平无明显差异性。 2.体外实验 2.1在体外使用缺氧条件（1%O2浓度、5%CO2浓度）培养HMEC-1（人类微血管内皮细胞），其中一组为单纯缺氧组（H组），另一组使用药物G6976预处理细胞30min后再缺氧培养（H+G组），均以常氧组（21%O2浓度、5%CO2浓度）为对照组。 2.2缺氧或药物刺激12h后，各组细胞在缺氧条件下无论是否添加磷酸化酶抑制剂G6976干预，Western blot检测结果表明HIF-1α和VEGFa蛋白均高表达。在各组当中HDAC4总蛋白表达水平无明显差异性，但P-HDAC4632蛋白与HIF-1α和VEGFa蛋白表达情况并不一致，H组P-HDAC4632明显高表达于常氧组，而H+G组P-HDAC4632蛋白表达减少。 2.3实时荧光定量PCR检测结果表明，相比常氧对照组，单纯缺氧组RCAN2基因明显高表达；然而使用磷酸化酶抑制剂G6976预处理之后，RCAN2基因表达水平明显减少。 2.4相比较于常氧组，H组HMEC-1细胞成管能力明显增强；而H+G组细胞成管受到限制。 结论： 本研究的体内脑缺血动物模型实验结果显示，脑缺血后缺血皮质区微血管密度增多，缺血皮质区HIF-1α和VEGFa表达明显上调，同时伴随HDAC4蛋白磷酸化水平的明显变化；进一步通过体外血管内皮细胞缺氧实验证实，缺氧会激活血管内皮细胞的HIF-VEGF信号通路，同时还明显影响血管内皮细胞的HDAC4磷酸化水平；磷酸化酶抑制剂G6976可显著抑制HDAC4的磷酸化，并抑制内皮细胞中VEGF下游成血管基因RCAN2的表达及其成管能力。研究结果表明，HDAC4蛋白磷酸化水平在脑组织缺血缺氧后血管新生过程中可能发挥重要调控作用，其可能是HIF-VEGF信号通路调控血管新生的重要靶点。
[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
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
本文编号：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
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.3
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本文编号：2495891
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