CRFR1参与低氧诱导的星形胶质细胞水肿

发布时间：2019-05-18 04:25

【摘要】：低氧作为一种非特异性应激原可诱导机体产生应激反应。哺乳动物通过下丘脑-垂体-肾上腺皮质轴(Hypothalamus-pituitary-adrenal axis, HPA)对机体面临的各种应激做出应答反应。应激促进下丘脑室旁核(Paraventricular nucleus, PVN)神经元释放下丘脑促肾上腺皮质激素释放激素(Corticotropin releasing factor, CRF)。 CRF主要通过和促肾上腺皮质激素释放激素I型受体(Corticotropin releasing factor receptor-1, CRFR1)和促肾上腺皮质激素释放激素II型受体(Corticotropin releasing factor receptor-2, CRFR2)结合发挥生理作用,参与神经和体液调节。环境低氧严重时可导致机体发生脑水肿,脑水肿的发生、发展与水通道蛋白(Aquaporins, AQPs)密切相关。AQPs是细胞膜上一类特异性水通道蛋白,参与多种类形细胞的水分子跨膜运输。其中水通道蛋白4(Aquaporin-4, AQP4)在哺乳动物脑组织中分布最为丰富,在正常生理状态下参与大脑水平衡调节,在病理损伤时则参与脑水肿形成和消退。AQP4在星形胶质细胞中大量表达,因此星形胶质细胞的肿胀与脑水肿有重要关系。低氧诱导因子-1(Hypoxia induced factor-1, HIF-1)是低氧敏感因子,参与调节机体氧平衡。低氧上调HIF-la蛋白表达,使HIF-1稳定性增加并与低氧反应元件(Hypoxia response element, HRE)结合作用下游靶基因,调控生理或病理作用。HIF-1调控的靶基因有血管内皮生长因子(Vascular endothelial growth factor, VEGF)及其受体(Vascular endothelial growth factor receptor, VEGFR)、诱导型一氧化氮合酶(Inducible nitric oxide synthase, iNOS)等。iNOS是一氧化氮合酶(Nitric oxide synthase, NOS)的一种,主要参与机体免疫反应,能够催化L-精氨酸(L-Arginine)产生一氧化氮(Nitric oxide, NO)。 NO是著名的气体信号分子,可以通过NO-cGMP-PKG通路调节细胞和机体功能。CRFR1在中枢神经系统和外周系统广泛分布,在星形胶质细胞中也有表达。我们实验室前期结果表明,低氧时下丘脑CRF分泌增加,前额叶皮层CRF基因转录升高。因此,我们设想机体在严重低氧时一方面通过CRF释放增加,激活星形胶质细胞CRFR1,引发胞内信号通路；另一方面细胞在严重低氧时胞内低氧诱导因子-1α(Hypoxia induced factor-1α, HIF-1α)激活,驱动下游信号通路。这两条通路共同作用于AQP4,增加AQP4水通道功能。本研究通过低氧培养箱模拟星形胶质细胞低氧环境,通过western blot、荧光标记法和细胞免疫荧光等方法,对CRFR1如何参与低氧诱导的星形胶质细胞水肿进行了研究。研究结果发现AQP4与CRFR1在大鼠原代皮层星形胶质细胞共表达；10nM和100nM CRF均能诱导大鼠原代星形胶质细胞胞内钙离子浓度升高,该作用可以被预孵CRFR1拮抗剂CP154,526阻断。同时发现大鼠原代星形胶质细胞在低氧(1%02)下HIF-1α蛋白表达增加,但并不表达iNOS蛋白；低氧上调大鼠原代小胶质细胞iNOS表达。这些结果提示低氧诱导皮层神经元分泌CRF,通过在星形胶质细胞G-蛋白偶联受体CRFR1激活胞内第二信使Ca2+,增加AQP4水通透性；低氧使胞内HIF-1α蛋白集聚增多,诱导下游低氧相关靶基因表达,促进AQP4水通透性增加。创新点： 1.发现低氧不激活离体培养大鼠原代星形胶质细胞的iNOS-NO-PKG通路。
[Abstract]:Hypoxia, as a non-specific stress, can induce stress reaction in that body. The mammal responds to the various stresses facing the body through the hypothalamic-pituitary-adrenal axis (HPA). Stress promotes the release of the corticotropin-releasing hormone (CRF) from the paraventricular nucleus (PVN) of the hypothalamus. The CRF mainly plays a physiological role in combination with the corticotropin-releasing hormone type I receptor (CRFR1) and the corticotropin-releasing hormone type II receptor (CRFR2), and is involved in the regulation of nerve and body fluid. The development of water channel protein (AQPs) is closely related to the development of water channel protein (AQPs). AAQPs is a class of specific water-channel proteins on the cell membrane, which is involved in the cross-membrane transport of water molecules in multi-species cells. The water channel protein 4 (Aquaporin-4, AQP4) is most abundant in the brain of the mammal, and is involved in the brain water balance regulation in the normal physiological state, and is involved in the formation and elimination of the brain edema in the case of pathological injury. The expression of AQP4 in astrocyte is a significant effect on the swelling and brain edema of astrocytes. The hypoxia-inducible factor-1 (HIF-1) is a hypoxia-sensitive factor and is involved in the regulation of the oxygen level in the body. The expression of HIF-la protein is up-regulated by hypoxia, and the stability of HIF-1 is increased and the downstream target gene is combined with the hypoxia response element (HRE) to control the physiological or pathological changes. The target gene regulated by. HIF-1 has vascular endothelial growth factor (VEGF) and its receptor (VEGFR) and inducible nitric oxide synthase (iNOS). The iNOS is a kind of nitric oxide synthase (NOS), which is mainly involved in the immune response of the organism and can catalyze the production of nitric oxide (NO) in L-arginine (L-Arginine). ). NO is a well-known gas signal molecule that can be used to regulate cell and body work through the NO-cGMP-PKG pathway can. CRFR1 is widely distributed in the central nervous system and the peripheral system, and there are also tables in astrocyte Da. The early results of our laboratory indicated that the secretion of CRF in the hypothalamus of the prefrontal cortex increased, and the transcription of CRF gene in the prefrontal cortex was increased. High. Therefore, we imagine that, on the one hand, in the case of severe hypoxemia, on the one hand, the increase of CRFR1 of the astrocytes is activated and the intracellular signaling pathway is induced; on the other hand, the cells are activated by the hypoxemia induction factor-1 (HIF-1) in severe hypoxia, and the downstream signal is driven. Road. These two paths act together on AQP4 and increase AQP4 water channel work In this study, the hypoxia environment of astrocyte was simulated by a low-oxygen incubator, and the method of western blot, fluorescent labeling and cell immunofluorescence was used to study how CRFR1 was involved in hypoxia-induced astrocyte edema. The results of the study showed that AQP4 and CRFR1 were co-expressed in primary rat cortical astrocytes; both 10 nM and 100 nM CRF were able to induce an increase in intracellular calcium ion concentration in primary astrocytes of the rat, which could be pre-treated with CRFR1 antagonists CP154,52, 6. At the same time, the expression of HIF-1 was increased under hypoxia (1%02), but the iNOS protein was not expressed. S expression. These results suggest that the hypoxia-induced cortical neurons secrete CRF and increase the water permeability of AQP4 by activating the second messenger Ca2 + in the intracellular second messenger Ca2 + in the G-protein-coupled receptor CRFR1 of the astrocyte. To promote AQP4 water permeability due to expression The sex is increased. Innovation point:1. It is found that hypoxia does not activate the iNOS-N in the primary astrocytes of the isolated rat
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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