AMPK通过mTOR促进脑缺氧条件下血管内皮细胞作用的研究

发布时间：2018-01-20 10:17

本文关键词： 脑血管内皮细胞乏氧腺苷酸活化蛋白激酶雷帕霉素靶蛋白自噬　出处：《郑州大学》2016年博士论文　论文类型：学位论文

【摘要】：背景与目的大脑的是高血流量、高耗氧量的器官,脑的重量仅占体重的2%,血流量却占心输出量的15%左右,耗氧量约占全身耗氧量的20%。但同时,大脑的能源储备极少,几乎只能利用脑循环提供的氧和葡萄糖进行有氧代谢,对脑循环有高度的依赖性。一旦发生完全性缺血缺氧超过5分钟,脑细胞就会发生不可逆性死亡,所以脑循环功能的正常意义重大,尤其是在急性脑梗死等缺血缺氧条件下,脑循环功能决定了脑部病变的发展和转归。脑循环是一个高度专业化的血管床,脑血管内皮细胞在脑循环的调节中起着重要的作用。脑血管内皮细胞在一些病理情况下如缺血缺氧时,经常会受到损伤,我们以往对脑缺血缺氧的研究都主要集中在神经元、神经胶质细胞、神经干细胞等方面,对脑血管内皮细胞的研究不多。除了常见的脑血管病以外,许多脑部疾病均伴发有缺血缺氧,所以研究脑血管内皮细胞缺血缺氧时的功能改变及其机制有重要的临床意义。缺氧时,脑血管内皮细胞的功能改变主要为：以增殖、凋亡、自噬等为表现的细胞生存改变和内分泌变化,而生存改变是基础,所以本实验主要研究缺氧时血管内皮细胞的增殖、凋亡和自噬的变化,并探讨其机制。腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)是代谢的感应器,哺乳动物AMPK是由三个亚基组成的异三聚体复合体,包括α亚基、p调控亚基和γ亚基。a催化亚基含有一个高度保守的丝氨酸苏氨酸激酶结构域,靠近活化环的N-末端,Thr-172磷酸化对酶的活性起关键作用；p亚基没有催化活性,可能与细胞内AMPKαβγ复杂体装配和细胞传感糖原相关；γ亚基通过感知细胞内ATP、ADP和AMP的浓度调节酶的活性。通过激活AMPK保持能量的平衡代谢途径(ATP生产)和抑制合成代谢途径(ATP消耗)。AMPK在营养条件差条件下激活,而营养丰富的条件下失去活性。AMPK激活下游的效应包括脂质和糖代谢、能量消耗、免疫反应、细胞生长和极性。AMPK广泛存在于人体多种组织,但在脑血管内皮细胞的研究不多。哺乳动物雷帕霉素靶蛋白(Mammalian target of rapamycin, mTOR)是调节细胞生长的一种丝氨酸苏氨酸激酶。在AMPK激活后mTOR活性抑制。mTOR活化都需要来自营养成分(葡萄糖,氨基酸)和生长因子的积极信号。在哺乳动物中,mTORC1复合物(mTOR complex 1, mTORC1)是由四个已知亚基构成：Raptor、PRAS40、mLST8和mTOR。mTORC1对营养信号敏感,可被细菌的大环内酯类雷帕霉素很快抑制,控制细胞的生长、血管生成及代谢。相反,mTORC2对营养信号是不敏感的,也不能被雷帕霉素很快抑制。mTORC1的两个已知底物是4EBP1和p70核糖体S6激酶。除了这些生长刺激信号激活]mTORC1,复合体可通过各种细胞应激迅速失活,从而保证细胞不受损害。mTORC1复合体的一个独特的方面是,它是依赖于养分利用而表现激酶活性。在低氧气条件下,细胞激活一系列关键通路而适应生存,其中大多数途径的是由转录因子缺氧诱导因子1(Hypoxia inducible factor 1,HIF-1)调节的,这在很大程度上依赖于细胞利用氧的能力作为信号来控制]HIF-1α的稳定性和转录活性。活化的HIF-1是由HIF-1α和HIF-1β两亚基组成构成异源二聚体,HIF-1α受氧浓度的精密调节且不稳定,亚基与氧水平无关。HIF-1β是乏氧诱导反应HIF-1α的关键分子,乏氧条件下,它被诱导表达。以前的研究证明在神经系统发挥重要作用,但在脑血管内皮细胞的作AMPK用还知之甚少,本课题我们将用分离的小鼠脑血管内皮细胞研究乏氧条件下在脑血管内皮细胞的功能,包括脑血管内皮细胞的增殖和凋亡,并研究其AMPK功能是否通过HIF-1进行的。自噬是细胞维持其细胞稳态的一个重要机制,我们将研究在脑血管内皮细胞自噬中的作用及机制。AMPK-mTOR第一部分乏氧条件-下在脑血管内皮细胞增殖及介导线粒体途AMPK径凋亡中的作用目的：研究脑血管内细胞在乏氧条件下表达的情况。明确HIF-1是否AMPK参与了乏氧条件下的活化。研究乏氧条件下AMPK对血管内皮细胞增殖、AMPK凋亡的作用及机制。方法：首先分离出小鼠脑血管内皮细胞(Cerebrovascular endothelial cells, CVEC),然后将其在乏氧(1%02)和常氧条件下进行培养,分别观察乏氧0、4、12和24小时的MPK表达,用Real time PCR法检测AMPK的表达。为明确HIF-1是否参与了乏氧条件下AMPK的活化,我们将小鼠血管内皮细胞在乏氧条件下培养,并用HIF-1α抑制剂处理细胞,检测细胞的AMPK活性。研究AMPK在脑血管内皮细胞中的增殖和凋亡的作用,用MTT法检测血管内皮细胞在乏氧条件下的增殖,并用AMPK shRNA及AMPK抑制剂下调AMPK后观察血管内皮细胞的增殖。用流式细胞仪检测乏氧条件下血管内皮细胞的凋亡率,并用AMPK shRNA下调AMPK后观察血管内皮细胞的凋亡率。同时用Western blot法检测脑血管内皮细胞中Bcl-2、Cl-caspase3的表达,用活性氧检测试剂盒检测乏氧下下调AMPK后脑血管内皮细胞ROS的产生量。结果：我们发现在小鼠脑血管内皮细胞主要表达AMPKα2,且磷酸化AMPK在乏氧条件下增加。当抑HIF-1α后,磷酸化的AMPK下降,同时AMPK也下降,证明乏氧能促进HIF-1α表达,能促进AMPK的活化。AMPK能促进细胞增殖。AMPK能抑制细胞凋亡,可能是通过抑制caspase3和促进Bcl-2实现的。干扰AMPK能增加线粒体ROS的产生量。第二部分乏氧条件下AMPK-mTOR调节脑血管内皮细胞自噬目的：观察乏氧时脑血管内皮细胞的自噬情况。研究乏氧促进细胞自噬是通过HIF-1进行的；研究AMPK与细胞凋亡的关系。研究mTOR对血管内皮细胞的作用及是否受AMPK调控,并初步探讨乏氧时脑血管内皮中凋亡和自噬的关系。方法：将CMEC进行乏氧处理,用免疫荧光观察LC3蛋白的分布,Western blot检测乏氧条件下CMEC的LC3 I、LC3 II亚型蛋白表达,并观察下调AMPK后期的表达情况。为研究乏氧促进细胞自噬是通过HIF-1进行的,脑血管内皮细胞用HIF-1的抑制剂处理,用Westeren Blot检HIF-1α磷酸化AMPK、LC3 Ⅰ、LC3 Ⅱ亚型蛋白的表达。用免疫沉淀检测下调AMPK后,脑血管内皮细胞中Bcl-2和Beclin的表达。为了研究AMPK与细胞凋亡的关系,我们检测了在AMPK下调的脑血管内皮细胞caspase-8活性及Bid的表达。为研究mTOR对血管内皮细胞的作用及是否受AMPK调控,我们将乏氧培养的脑血管内皮细胞AMPK下调,Westernblot观察脑血管内皮细胞中AMPK、mTOR、P-mTOR、S6K、P-S6K的表达；为进一步研究mTOR、AMPK和自噬之间的关系,我们用shRNA方法处理脑血管内皮细胞,Western blot检测mTOR、LC3表达,用免疫荧光观察细胞内LC3的分布,免疫沉淀检测脑血管内皮细胞中Bcl-2和Beclin的表达。结果：乏氧时,LC3蛋白重新分配到不同的点,说明细胞正经历自噬,而对照组细胞则散布于细胞内,Western blot显示乏氧条件下CMEC的LC3 II亚型蛋白水平增加,下调AMPK后,LC3 II亚型蛋白水平下降,证明乏氧条件下血管内皮细胞自噬增加。HIF-1被抑制后磷酸化AMPK显著降低,说明HIF-1可能通过活化AMPK诱导细胞自噬。乏氧时,对照组血管内皮细胞有较低的水平capase-8活性,而在AMPK下调细胞caspase-8活性增加,Bid表达增加,说明乏氧条件下,AMPK抑制脑血管内皮细胞凋亡。下调脑血管内皮细胞AMPK后,P-S6K水平增加,但总mTOR、S6K水平没有明显改变,说明AMPK抑制后,mTOR、S6K激活；抑制：mTOR表达后,CMEC的LC3 II亚型蛋白水平增加,mTOR表达下调细胞免疫荧光LC3染色,结果表明mTOR抑制后,细胞自噬能力增强,证明了乏氧条件下,mTOR抑制脑血管内皮细胞自噬。同时发现在mTOR表达下调细胞Bcl-2显著增加,证明乏氧时,脑血管内皮细胞自噬增加后抑制凋亡。结论本课题证明了乏氧条件下,AMPK-mTOR有促进脑血管内皮细胞增殖、抑制其凋亡和促进其自噬的功能,并探讨了其分子机制,为临床治疗脑血管相关的疾病奠定了一定的理论基础。
[Abstract]:Background and purpose of the brain is high blood flow, high oxygen consumption of the organs, the brain weight of only 2% of the body weight, blood flow and cardiac output accounted for about 15%, the oxygen consumption of the body about the oxygen consumption of 20%. but at the same time, the brain's energy reserves are almost only by providing oxygen and glucose of cerebral circulation aerobic metabolism, is highly dependent on cerebral circulation. Once complete cerebral ischemia occurred more than 5 minutes, brain cells would be an irreversible death, so the normal function of cerebral circulation is significant, especially in acute cerebral infarction and ischemic hypoxia, cerebral circulation function determines the development of the brain lesions and prognosis. The cerebral circulation is a highly specialized vascular bed, cerebral vascular endothelial cells in the brain plays an important role in the regulation of cerebral circulation. Vascular endothelial cells in some pathological conditions such as ischemia and hypoxia, often due to injury, Our previous study on cerebral ischemia and hypoxia are mainly in neurons, glial cells, neural stem cells, not much research on cerebral vascular endothelial cells. In addition to common cerebrovascular disease, many brain diseases are associated with ischemia and hypoxia, has important clinical significance and mechanism of hypoxia so function change the cerebral vascular endothelial cells during ischemia. The changes of cerebral hypoxia, vascular endothelial cell function mainly: proliferation, apoptosis, autophagy is the cell survival and endocrine change change, and change is the basis for survival, so this experiment of hypoxia on the proliferation of vascular endothelial cells, apoptosis and autophagy, and to explore its mechanism. Amp activated protein kinase (AMP-activated protein kinase, AMPK) is a metabolic sensor, the mammalian AMPK is ISO three dimer complex composed of three subunits, package The alpha subunit, P regulatory subunit gamma subunit.A and catalytic subunit containing a highly conserved serine threonine kinase domain, near the end of N- activation loop, Thr-172 phosphorylation on enzyme activity plays a key role; the P subunit has no catalytic activity, and intracellular AMPK alpha beta gamma complex the assembly and cell glycogen sensing gamma subunit; by sensing the intracellular ATP concentration of ADP and AMP in the regulation of enzyme activity. To keep the balance of energy metabolism through the activation of the AMPK pathway (ATP production) and inhibit the synthesis of metabolic pathways (ATP consumption) under the conditions of activation of.AMPK in poor nutritional conditions, effects of nutrient rich conditions loss of activity of.AMPK activated downstream including lipid and glucose metabolism, energy consumption, immune response, cell growth and polarity of.AMPK exists widely in human tissues, but the study of cerebral vascular endothelial cells is the mammalian target of rapamycin (Mam. Malian target of rapamycin, mTOR) is a serine threonine kinase regulates cell growth. The activation of AMPK mTOR activity and inhibit the activation of.MTOR needs to come from the nutrients (glucose, amino acids) and growth factor positive signal. In mammals, the mTORC1 complex (mTOR complex 1, mTORC1) is composed of four known subunits: Raptor, PRAS40, mLST8 and mTOR.mTORC1 are more sensitive to nutritional signals can be macrolide rapamycin bacteria quickly suppressed, control of cell growth, angiogenesis and metabolism. On the contrary, mTORC2 is not sensitive to nutrient signal, can not be the two known substrate inhibition of.MTORC1 is rapamycin soon 4EBP1 and p70 ribosomal S6 kinase. In addition to these growth signals to activate the]mTORC1 complex, through a variety of cellular stress rapidly inactivated, so as to ensure the cell from being damaged by a unique.MTORC1 complex The aspect is that it is dependent on nutrient utilization and performance of the kinase activity. In low oxygen conditions, cells activate a series of key pathways and adapt to survive, most of the way by the transcription factor hypoxia inducible factor 1 (Hypoxia inducible 1 factor, HIF-1) regulation, which depends on the stability and transcriptional activity in cells with oxygen the ability to control the]HIF-1 alpha as a signal to a great extent. The activation of HIF-1 is composed of HIF-1 alpha and HIF-1 beta two subunit composition of two heterologous dimers, HIF-1 alpha by oxygen concentration of fine adjustment and unstable, subunits and oxygen levels without.HIF-1 beta is a key molecule of hypoxia induced reaction of HIF-1 alpha, under hypoxic conditions, it is induced. Previous studies have shown that play an important role in the nervous system, but in the cerebral vascular endothelial cells AMPK with very little is known about this topic, we will use the separation of murine brain microvascular endothelial cells In the study of cerebral vascular endothelial cells under hypoxic conditions, including the proliferation and apoptosis of cerebral vascular endothelial cells, and to study the function of AMPK is carried out through the HIF-1. Autophagy is an important mechanism for the cell to maintain cell homeostasis, we will study the role and mechanism of.AMPK-mTOR in cerebral vascular endothelial cells autophagy in the first part under hypoxic conditions in cerebral vascular endothelial cells proliferation and induced mitochondria mediated apoptosis. AMPK pathway Objective: To study the expression of cerebral vascular cells in hypoxic conditions. To determine whether HIF-1 is involved in the activation of AMPK under hypoxic conditions. The study of AMPK under hypoxic conditions on the proliferation of vascular endothelial cell function AMPK and the mechanism of apoptosis. Methods: first isolated from murine brain microvascular endothelial cells (Cerebrovascular endothelial cells, CVEC), and then in hypoxia (1%02) and cultured under normoxic conditions, respectively. The observation of hypoxic 0,4,12 and 24 hours of MPK expression, used to detect the expression of AMPK Real time PCR. To determine whether HIF-1 is involved in the activation of AMPK under hypoxic conditions, we cultured vascular endothelial cells in mice under hypoxic conditions, and using the HIF-1 alpha inhibitor treated cells, the activity of AMPK cells was detected. AMPK the proliferation and apoptosis in cerebral vascular endothelial cells in the role of proliferation was detected by MTT in vascular endothelial cells under hypoxic conditions, to observe the proliferation of vascular endothelial cells by AMPK shRNA and AMPK inhibitor down regulated after AMPK. Flow cytometry was used to detect the apoptosis of vascular endothelial cells lack oxygen conditions, and AMPK shRNA AMPK was observed after downregulation of vascular endothelial cell apoptosis rate. At the same time with Western blot method to detect the cerebral vascular endothelial cells in Bcl-2, Cl-caspase3 expression by reactive oxygen detection reagent box under hypoxia reduced cerebral vascular AMPK Production of skin cell ROS. Results: we found that in mice cerebral vascular endothelial cells express AMPK alpha 2 and phosphorylated AMPK increased under hypoxic condition. When the anti HIF-1 alpha, phosphorylation of AMPK decreased, while AMPK decreased, that hypoxia can promote HIF-1 expression, promote AMPK the activation of.AMPK can promote the proliferation of.AMPK cells can inhibit cell apoptosis, possibly through inhibition of Caspase3 and promote the implementation of Bcl-2. AMPK can increase the amount of interference generated in mitochondrial ROS. The second part under hypoxic conditions AMPK-mTOR regulates autophagy in cerebral vascular endothelial cells Objective: cerebral vascular endothelial cell autophagy was observed in hypoxia. Hypoxia promotes research autophagy is carried out through the HIF-1; to study the relationship between AMPK and apoptosis. The effect of mTOR on vascular endothelial cells and is regulated by AMPK, and to investigate the apoptosis and autophagy of vascular endothelium of brain hypoxia . methods: CMEC for hypoxia treatment, observe the distribution of LC3 protein by immunofluorescence and Western blot detection of CMEC under hypoxic conditions by LC3 I, the expression of LC3 subtypes of II protein, and to observe the expression of AMPK down later. To study hypoxia promotes cell autophagy is carried out through the HIF-1, cerebral vascular endothelial cells treated with the inhibitors of HIF-1, Westeren Blot HIF-1 alpha phosphorylation of AMPK, LC3 I, the expression of LC3 II isoforms. Detection of down-regulation of AMPK by immunoprecipitation, the expression of Bcl-2 and Beclin in cerebral vascular endothelial cells. To study the relationship between AMPK and apoptosis, we examined the expression of AMPK in down cerebral vascular endothelial cells caspase-8 activity and Bid. To study the effects of mTOR on vascular endothelial cells and is regulated by AMPK, cerebral vascular endothelial cells under hypoxia AMPK we will cut, Westernblot observation of cerebral vascular endothelial cells in AMP K, mTOR, P-mTOR, S6K, P-S6K expression; for the further study of mTOR, the relationship between AMPK and autophagy, we treat cerebral vascular endothelial cells by shRNA method, Western blot detection of mTOR, LC3 expression, observe the distribution of LC3 in cells by immunofluorescence and immunoprecipitation to detect the expression of Bcl-2 and Beclin in cerebral vascular endothelial cells the results: hypoxia, LC3 protein re assigned to different points, indicating that cells undergoing autophagy, whereas the control group cells were scattered in the cytoplasm, Western blot showed that under hypoxic conditions CMEC LC3 subtype II protein level increased, decreased AMPK, LC3 subtype II protein level decreased, that under hypoxic conditions of vascular endothelial cells autophagy increased phosphorylation of AMPK.HIF-1 was inhibited significantly reduced, indicating that HIF-1 may through the activation of AMPK induced autophagy. The hypoxia control group, vascular endothelial cells have lower levels of capase-8 activity, whereas in AMPK To increase the activity of caspase-8 cells, increase the expression of Bid, that under hypoxic conditions, AMPK inhibited the apoptosis of cerebral vascular endothelial cells. Downregulation of cerebral vascular endothelial cells after AMPK, P-S6K levels increased, but the total mTOR did not change significantly the level of S6K, indicating that AMPK inhibited after mTOR, S6K activation; inhibition: the expression of mTOR, CMEC LC3 subtype II protein level increased mTOR expression by immunofluorescence LC3 staining, the results showed that mTOR inhibited, enhanced autophagy capacity, proved that under hypoxic conditions mTOR inhibition of cerebral vascular endothelial cells autophagy. At the same time found in mTOR expression Bcl-2 cells increased significantly, that of hypoxia, cerebral vascular endothelial cells autophagy increased after inhibition of apoptosis. Conclusion this study proved that under hypoxic conditions, AMPK-mTOR can promote the proliferation of vascular endothelial cells, inhibit apoptosis and promote the function of autophagy, and explore its molecular mechanism for the clinical treatment. It lays a theoretical basis for cerebrovascular diseases.

【学位授予单位】：郑州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R743

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