内皮源性NO介导的低氧信号启动星形胶质细胞低氧代偿的机制研究

发布时间：2018-07-24 13:03

【摘要】：目的:大脑对缺氧最为敏感,缺氧可导致脑水肿和神经细胞受损,严重影响大脑功能。对于不同程度的乏氧性缺氧(hypoxic hypoxia),大脑将表现出不同程度的神经功能紊乱,如急性重度缺氧将引起头痛、烦躁、惊厥、昏迷甚至死亡;慢性中度缺氧将引起疲劳、嗜睡、注意力不集中、记忆力下降等症状;而轻度及早期乏氧性缺氧发生时由于脑血管及星形胶质细胞系统的代偿性低氧保护反应,可为神经元提供充分的氧供和能量底物,以维持大脑的正常生理功能,因此,研究轻度缺氧条件下中枢神经系统(central nervous system,CNS)代偿性低氧保护机制,对于乏氧性缺氧脑损伤疾病的预防和治疗具有重要意义。CNS结构与功能错综复杂,神经血管单元(neurovascular units,NVU)是其最小结构与功能单位,该模型主要由脑微血管、星形胶质细胞和神经元共同构造组成。星形胶质细胞处于NVU物质转运与信息传递的中心,充当神经元与微血管之间的纽带与桥梁作用。当星形胶质细胞感受到氧供减少后,能够启动代偿性低氧保护反应,包括减少葡萄糖有氧氧化、增加无氧糖酵解,降低自身氧耗、促进葡萄糖与乳酸转运、释放血管新生因子等,是维持神经元正常生理功能的重要代偿机制。根据NVU结构的特点,脑微血管内皮细胞(brain microvascular endothelial cell,BMEC)紧临血液,可最先感知血氧分压降低,其后才是星形胶质细胞和神经元。因此如能在星形胶质细胞感受到氧供减少前启动代偿性低氧保护反应,对于维持神经元细胞的正常生理功能无疑具有更为重要的生理意义。在缺氧发生时,BMEC最先感受到血氧分压降低,并引起一系列的血管系统代偿性低氧保护反应,而最重要也是最快速的代偿方式是通过舒张血管的作用增加局部组织供氧量,此过程是内皮细胞释放一氧化氮(nitric oxide,NO),并扩散至血管平滑肌细胞,通过活化可溶性鸟苷酸环化酶(s GC)的方式启动舒张血管作用,而内皮细胞释放的NO能否继续扩散至其他邻近细胞,并发挥相应的生理功能与效应,有待进一步探索。NO是重要的细胞信使分子,参与机体多种生理或病理过程,在机体心血管系统、神经系统和免疫系统中发挥重要的生物学作用。研究表明NO除了具有舒张血管的作用外,还参与低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)的稳定性调控,通过干预脯氨酰羟化酶(PHD)绑定Fe2+而阻断PHD酶的活性,从而阻断HIF-1α的降解,还可通过PI3K/Akt/m TOR信号通路诱导HIF-1α表达上调。鉴于NO分子可调控HIF-1α稳定性与表达水平,并且是重要的内源性细胞信使分子,无疑是理想的低氧信号传导分子,因此我们将NO分子作为重要的非氧气的调节因素与信息分子进行低氧信号传导的研究对象。既然轻度缺氧时,BMEC释放的NO能够作为低氧信号扩散至血管平滑肌细胞产生舒张血管的作用,那么BMEC释放的NO能否继续扩散至邻近的星形胶质细胞,并在其未感知缺氧时通过HIF-1α启动代偿性低氧保护反应?有待研究证实。基于上述分析,本研究拟分离培养大鼠原代脑星形胶质细胞和原代BMEC,探索脑星形胶质细胞未感受缺氧的氧气阈值浓度,并在此缺氧条件下研究外源性给药产生和共培养BMEC源性释放的NO预处理能否阻断脑星形胶质细胞HIF-1α蛋白的降解,以及对葡萄糖代谢与无氧糖酵解、葡萄糖和乳酸转运、血管新生等相关基因的转录和表达调控;同时对照清除NO或抑制NO生成,并结合运用RNAi技术干扰HIF-1α基因转录,探索NO-低氧信号如何介导脑星形胶质细胞的低氧保护反应,以期初步阐明BMEC源性NO介导的低氧信号传导启动脑星形胶质细胞低氧保护反应的作用机制,为进一步研究乏氧性缺氧脑损伤疾病的致病机制与干预保护措施奠定基础。方法:第一部分大鼠脑星形胶质细胞、脑微血管内皮细胞的原代分离培养与鉴定1.分离培养原代脑星形胶质细胞及细胞鉴定:采用一步酶消化及振荡纯化的方法分离培养大鼠大脑星形胶质细胞;免疫荧光染色法检测GFAP的表达用于脑星形胶质细胞的鉴定;2.分离培养原代脑微血管内皮细胞及细胞鉴定:采用两步酶消化、梯度密度离心及嘌呤霉素纯化的方法分离培养大鼠大脑微血管内皮细胞;免疫荧光染色法检测v WF相关抗原的表达用于脑微血管内皮细胞的鉴定。第二部分外源性NO启动轻度缺氧星形胶质细胞低氧保护反应的实验研究1.不同氧气浓度条件对原代脑星形胶质细胞HIF-1α蛋白水平的影响:Western blot检测21%、9%、7%、5%、3%和1%氧气浓度条件下星形胶质细胞HIF-1α蛋白水平,以获得星形胶质细胞未感受缺氧的氧气阈值浓度;2.5%O_2轻度缺氧条件下,DETA(NO供体)对原代脑星形胶质细胞HIF-1α蛋白水平的影响:Western blot检测DETA不同剂量处理和不同处理时间后脑星形胶质细胞HIF-1α蛋白水平;3.5%O_2轻度缺氧条件下,DETA对原代脑星形胶质细胞葡萄糖代谢相关酶基因、葡萄糖和乳酸转运体蛋白基因、血管内皮生长因子(VEGF)基因转录水平,VEGF蛋白表达水平和乳酸(LAC)释放水平的影响:RT-q PCR检测DETA处理后脑星形胶质细胞PDK1、LDHA、LDHB、HK1、HK2、GLUT1、GLUT3、MCT1、MCT2、MCT4、VEGF基因转录水平;Western blot检测DETA处理后脑星形胶质细胞VEGF蛋白水平,酶促动力学比色法检测培养液LAC释放水平;4.干扰原代脑星形胶质细胞HIF-1α基因转录后,DETA对5%O_2轻度缺氧条件下脑星形胶质细胞葡萄糖代谢相关酶基因、葡萄糖和乳酸转运体蛋白基因、VEGF基因转录水平,VEGF蛋白表达水平和LAC释放水平的影响:采用HIF-1α特异性si RNA干扰原代脑星形胶质细胞HIF-1α基因转录,RT-q PCR和Western blot鉴定细胞转染效率;RT-q PCR检测DETA处理后脑星形胶质细胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因转录水平;Western blot检测DETA处理后星形胶质细胞VEGF蛋白水平,酶促动力学比色法检测培养液LAC释放水平。第三部分脑微血管内皮细胞源性NO启动轻度缺氧星形胶质细胞低氧保护反应的实验研究1.5%O_2轻度缺氧条件下,原代脑微血管内皮细胞和原代脑星形胶质细胞NO的释放水平:荧光分析法分别检测脑微血管内皮细胞和脑星形胶质细胞培养液中Nitrite/Nitrate水平;2.5%O_2轻度缺氧条件下,脑微血管内皮细胞源性NO对原代脑星形胶质细胞HIF-1α蛋白水平的影响:采用Transwell培养板进行原代脑微血管内皮细胞与原代脑星形胶质细胞共培养,Western blot检测共培养后脑星形胶质细胞HIF-1α蛋白水平;3.5%O_2轻度缺氧条件下,脑微血管内皮细胞源性NO对原代脑星形胶质细胞葡萄糖代谢相关酶基因、葡萄糖和乳酸转运体蛋白基因、VEGF基因转录水平,VEGF蛋白表达水平和LAC释放水平的影响:采用Transwell培养板进行原代脑微血管内皮细胞与原代脑星形胶质细胞共培养;RT-q PCR检测脑星形胶质细胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因转录水平;Western blot检测原代脑星形胶质细胞VEGF蛋白水平;酶促动力学比色法检测培养液LAC释放水平。结果:第一部分:1.获得原代培养的大鼠大脑星形胶质细胞,纯度为98.4%;2.获得原代培养的大鼠脑微血管内皮细胞,纯度为95.2%。第二部分:1.与常氧条件(21%O_2)相比,1~3%氧气浓度条件下,原代脑星形胶质细胞HIF-1α蛋白水平显著升高,而5~9%氧气浓度条件下,HIF-1α蛋白水平无显著性变化,提示5%O_2氧气浓度条件是原代脑星形胶质细胞未感受缺氧的临界浓度,我们将此低氧条件定义本实验研究的轻度缺氧条件;2.5%O_2轻度缺氧条件下,1.0 m M DETA(NO供体)预处理12 h能够诱导原代脑星形胶质细胞HIF-1α蛋白水平上调,但能被NO清除剂抑制;3.5%O_2轻度缺氧条件下,DETA预处理后原代脑星形胶质细胞PDK1、LDHA、HK1、HK2、GLUT1、GLUT3、MCT4、VEGF基因转录水平显著升高,VEGF蛋白水平和LAC释放水平显著升高;4.5%O_2轻度缺氧条件下,干扰原代脑星形胶质细胞HIF-1α基因转录能够抑制DETA上调PDK1、LDHA、HK1、HK2、GLUT1、GLUT3、MCT4、VEGF基因转录,抑制VEGF蛋白表达和LAC释放。第三部分:1.5%O_2轻度缺氧条件下,原代脑星形胶质细胞产生和释放的NO较少(86±41 n M Nitrite/Nitrate),而原代脑微血管内皮细胞能够释放一定量的NO(832±83 n M Nitrite/Nitrate,与0.8 m M DETA释放的Nitrite/Nitrate峰值浓度相当),但能被NOS抑制剂抑制;2.5%O_2轻度缺氧条件下,原代脑微血管内皮细胞与原代脑星形胶质细胞共培养后能够诱导脑星形胶质细胞HIF-1α蛋白水平的上调,但能被NOS抑制剂抑制;3.5%O_2轻度缺氧条件下,原代脑微血管内皮细胞与原代脑星形胶质细胞共培养后能够上调脑星形胶质细胞PDK1、LDHA、HK1、HK2、GLUT1、MCT4、VEGF基因转录,上调VEGF蛋白表达和LAC释放,但能被NOS抑制剂抑制。结论:在5%O_2轻度缺氧条件下,体外培养的原代大鼠脑星形胶质细胞未感受到低氧,而此低氧条件下给予NO供体药物和共培养脑微血管内皮细胞均能够使脑星形胶质细胞感受到低氧,并上调HIF-1α下游的与无氧糖酵解、葡萄糖和乳酸转运、血管新生等低氧代偿作用相关的酶或因子的基因转录或表达;对照清除NO或抑制NO生成,以及干扰HIF-1α基因转录的结果表明,NO通过阻断HIF-1α的降解,上调其下游的基因转录或表达。综上所述,轻度缺氧时原代大鼠脑微血管内皮细胞释放的NO可作为低氧信号,扩散传导至脑星形胶质细胞,并通过HIF-1α启动脑星形胶质细胞的低氧代偿性保护反应。本研究丰富了NO作为内源性细胞信号分子的效应机制,阐释了一种轻度缺氧时神经血管单元细胞间低氧信号的传导途径,为进一步研究乏氧性缺氧脑损伤疾病的致病机制与干预保护措施奠定了基础。
[Abstract]:Objective: the brain is most sensitive to hypoxia. Hypoxia can lead to brain edema and nerve cell damage, which seriously affect the brain function. For different degrees of hypoxia (hypoxic hypoxia), the brain will show different degrees of nerve dysfunction, such as acute severe anoxia, which will lead to headache, irritability, convulsion, coma and even death; chronic moderate deficiency. Oxygen will cause symptoms such as fatigue, lethargy, concentration of attention, and decline in memory; while mild and early anoxic hypoxia can provide sufficient oxygen supply and energy substrates for neurons to maintain normal physiological functions of the brain and study mild hypoxia at the time of hypoxic hypoxia. Under conditions, the compensatory hypoxic protection mechanism of the central nervous system (CNS) is of great significance for the prevention and treatment of hypoxic hypoxic brain damage. The.CNS structure and function are complex, and the neurovascular units (NVU) is the smallest structure and function unit. The model is mainly from the brain microvessel and the star. Astrocytes and neurons are constructed together. Astrocytes are located at the center of NVU material transport and information transfer, acting as a link between neurons and microvessels. When the astrocytes feel the oxygen supply is reduced, it can start compensatory hypoxic protection reverse, including reducing glucose oxygen oxidation and increasing no oxygen. Oxygen glycolysis, reduce oxygen consumption, promote the transport of glucose and lactic acid, release angiogenesis factors and so on. It is an important compensatory mechanism to maintain normal physiological function of neurons. According to the characteristics of NVU structure, brain microvascular endothelial cell (BMEC) is closely associated with blood. It is astrocytes and neurons. Therefore, it is undoubtedly of more important physiological significance to maintain the normal physiological function of neuron cells before the oxygen supply is reduced in astrocytes. In the case of hypoxia, BMEC first feels the reduction of blood oxygen pressure and causes a series of blood vessels. The system compensatory hypoxic protection reaction, and the most important and fastest way of compensation is the increase of local tissue oxygen supply through the role of diastolic blood vessels. This process is that endothelial cells release nitric oxide (nitric oxide, NO) and spread to vascular smooth muscle cells and activate diastolic blood by activating soluble guanosine cyclase (s GC). Whether or not the NO can continue to spread to other adjacent cells and play the corresponding physiological functions and effects, it is necessary to further explore.NO as an important cell messenger, participate in various physiological or pathological processes of the body, and play an important biological role in the body's cardiovascular system, the nervous system and the immune system. In addition to the effect of diastolic blood vessels, NO also participates in the regulation of the stability of the hypoxia inducible factor -1 alpha (hypoxia-inducible factor-1 a, HIF-1 alpha), blocking the activity of the PHD enzyme by interfering with the binding of Fe2+ to the prolyyl hydroxylase (PHD) and blocking the degradation of the HIF-1 alpha, and can also induce the expression of the HIF-1 alpha by PI3K/Akt/m TOR signal pathway. In view of the fact that NO molecules can regulate the stability and expression level of HIF-1 alpha and are important endogenous cell messenger molecules, it is undoubtedly an ideal hypoxia signal transduction molecule, so we use NO molecules as an important regulator of non oxygen and information molecules to carry out the study of hypoxia signal transduction. Since mild hypoxia, BMEC release NO can be used as a hypoxic signal to spread to vascular smooth muscle cells to produce diastolic blood vessels, then can the NO released by BMEC continue to spread to adjacent astrocytes and activate compensatory hypoxic protection by HIF-1 alpha in the absence of hypoxia? Primary astrocytes and primary BMEC were used to explore the oxygen threshold concentration in the brain astrocytes, and the study of NO preconditioning for exogenous drug delivery and co culture of BMEC derived release under this hypoxia condition could block the degradation of HIF-1 alpha protein in astrocytes, as well as glucose metabolism and anaerobic glycolysis, The transcriptional and expression regulation of glucose and lactic acid transport, angiogenesis and other related genes, simultaneously control NO or inhibit NO production, and combine with RNAi technology to interfere with HIF-1 alpha gene transcription, explore how NO- hypoxia signal mediates the hypoxic protective response of astrocytes in brain astrocytes, in order to clarify the hypoxia signal conduction mediated by BMEC derived NO. The mechanism of activating the hypoxic protective reaction of astrocytes in the brain was initiated to lay the foundation for further study on the pathogenesis and protective measures of hypoxic hypoxic brain injury. Methods: the first part of the rat brain astrocytes, the primary separation culture and identification of the cerebral microvascular endothelial cells were isolated and cultured for the isolation and culture of the primary astroglia. Cell and cell identification: the rat brain astrocytes were isolated and cultured with one step enzyme digestion and oscillatory purification. Immunofluorescence staining was used to detect the expression of GFAP in the identification of astrocytes. 2. the isolation and culture of primary cerebral microvascular endothelial cells and cell identification: two step enzyme digestion, gradient density centrifugation and purinamycin Purified rat brain microvascular endothelial cells were isolated and cultured; immunofluorescence staining was used to detect the expression of V WF related antigen in the identification of cerebral microvascular endothelial cells. Second the experimental study on the initiation of hypoxic protective reaction of mild hypoxic astrocytes by exogenous NO; 1. different oxygen concentration conditions for the primary astroglia Effect of cellular HIF-1 alpha protein level: Western blot detection of astrocytes HIF-1 alpha protein level in 21%, 9%, 7%, 5%, 3% and 1% oxygen concentrations to obtain oxygen threshold concentration in astrocytes without hypoxia; the effect of DETA (NO donor) on the level of HIF-1 a protein of primary astrocytes under mild 2.5%O_2: West Ern blot was used to detect the HIF-1 alpha protein level of astrocytes in DETA after different doses of treatment and different processing time; DETA gene, glucose and lactic acid transporter gene, VEGF gene transcription level, VEGF protein table under mild hypoxia in 3.5%O_2 RT-q PCR detection of PDK1, LDHA, LDHB, HK1, HK2, GLUT1, GLUT3, LDHB, GLUT1, GLUT3, RT-q PCR The effect of DETA on glucose metabolism related enzyme gene, glucose and lactic acid transporter gene, VEGF gene transcriptional level, VEGF protein expression level and LAC release level in the brain astrocytes of primary astrocytes after HIF-1 alpha gene transcription: HIF-1 alpha specific Si RNA interfering with the original brain astrocytes. HIF-1 alpha gene transcription of glial cells, RT-q PCR and Western blot identification of cell transfection efficiency, RT-q PCR detection of PDK1, LDHA, HK1, HK2, and transcriptional level after DETA treated astrocytes An experimental study on the initiation of hypoxic protective reaction of mild hypoxic astrocytes in the third part of cerebral microvascular endothelial cell derived NO, the release level of NO in primary cerebral microvascular endothelial cells and primary brain astrocytes under mild hypoxic conditions in 1.5%O_2: fluorescence analysis for the detection of cerebral microvascular endothelial cells and astrocytes respectively Nitrite/Nitrate level in the culture medium; the effect of NO on the level of HIF-1 alpha protein of primary brain astrocytes under mild hypoxic 2.5%O_2: Transwell culture plate was used to co culture the primary cerebral microvascular endothelial cells and the primary astrocytes, and the Western blot was used to detect the astrocytes after the culture of the brain astrocytes. The level of cell HIF-1 alpha protein; the effect of NO on glucose metabolism related enzyme gene, glucose and lactic acid transporter gene, VEGF gene transcription level, VEGF protein expression level and LAC release level of primary brain astrocytes under mild hypoxic condition of 3.5%O_2: using Transwell culture plate for primary generation Cerebral microvascular endothelial cells were co cultured with primary astrocytes; RT-q PCR was used to detect astrocytes PDK1, LDHA, HK1, HK2, GLUT1, MCT4, and VEGF transcriptional level; Western blot was used to detect the level of the primary astrocyte VEGF protein; enzyme kinetics colorimetric assay was used to detect the release level. Results: the first part: 1. obtained the original The purity of the cultured rat brain astrocytes was 98.4%; 2. obtained the primary cultured rat brain microvascular endothelial cells, and the purity was 95.2%. second: 1. compared with the normal oxygen condition (21%O_2), the level of HIF-1 alpha protein in the primary astrocytes was significantly increased under the 1~3% oxygen concentration condition, and the HIF-1 alpha protein under the 5~9% oxygen concentration condition. There is no significant change in level, suggesting that the 5%O_2 oxygen concentration condition is the critical concentration of the primary brain astrocytes in the absence of hypoxia. We define this hypoxic condition as a mild hypoxic condition. Under mild hypoxia, 1 m M DETA (NO donor) preconditioning 12 h can induce primary astrocyte HIF-1 alpha protein. The level was up, but could be suppressed by NO scavenger. Under mild hypoxia, 3.5%O_2, LDHA, HK1, HK2, GLUT1, GLUT3, MCT4, VEGF gene transcriptional level increased significantly after DETA pretreatment, and the level of VEGF protein and the release level increased significantly. Gene transcription can inhibit DETA up regulation of PDK1, LDHA, HK1, HK2, GLUT1, GLUT3, MCT4, VEGF gene transcription, inhibition of VEGF protein expression and LAC release. The third part: under the mild hypoxia conditions, the generation and release of primary brain astrocytes are less (86 + 41), and the primary cerebral microvascular endothelial cells can release a certain amount. NO (832 + 83 n M Nitrite/Nitrate, equivalent to the peak concentration of Nitrite/Nitrate released by 0.8 m M DETA), but can be inhibited by NOS inhibitors; under mild hypoxic condition, the primary brain microvascular endothelial cells co cultured with the primary astrocytes can induce the up regulation of astrocyte HIF-1 alpha protein level, but can be suppressed by NOS. Preparation inhibition; under mild hypoxic condition of 3.5%O_2, primary cerebral microvascular endothelial cells co cultured with primary astrocytes can up regulate astrocyte PDK1, LDHA, HK1, HK2, GLUT1, MCT4, VEGF gene transcription, up regulation of VEGF protein expression and LAC release, but can be inhibited by NOS inhibitor. Conclusion: under mild hypoxia conditions, in vitro, in vitro The cultured primary rat astrocytes did not feel hypoxic, and the NO donor drugs and co cultured brain microvascular endothelial cells under this hypoxic condition could make the brain astrocytes feel hypoxic, and up regulation of HIF-1 alpha downstream and anaerobic glycolysis, glucose and lactic acid transport, angiogenesis and other low oxygen compensatory effects The transcriptional or expression of the gene of the enzyme or factor; the results of eliminating NO or inhibiting NO production, and interfering with the transcription of HIF-1 a gene indicate that NO can up regulate the transcription or expression of the downstream genes by blocking the degradation of HIF-1 a. To sum up, the NO released by the primary rat cerebral microvascular endothelial cells in mild hypoxia can be used as a hypoxic signal and diffusion conduction. This study enriches the effect mechanism of NO as an endogenous cell signal molecule, and explains the conduction pathway of the hypoxia signal in the neurons of the neurovascular unit at the time of mild hypoxia, in order to further study the hypoxic hypoxic brain damage disease, and further study the disease of hypoxic hypoxic brain damage. The pathogenesis and intervention measures laid the foundation.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742

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