P2X7r调控AQP4表达在HBOP机制中的作用
发布时间：2019-06-22 11:49
【摘要】：背景和目的 脑缺血性疾病占脑血管疾病总数的90%以上，如何提高脑组织对缺氧的耐受，减轻缺血后神经及功能损伤，，一直是神经外科领域研究的难点和重点。本课题前期试验发现，高压氧预适应(hyperbaric oxygen preconditioning，HBOP)对脊髓损伤、脑缺血都表现出了积极的保护作用，但其具体作用机制尚不明确。 P2X7r（P2X7receptor）是ATP门控离子通道家族中的独特亚型，广泛分布于脑组织细胞，P2X7r作为上游调节位点，在神经系统参与多种功能活动，在缺血性脑损伤中发挥重要作用。AQP4是膜水通道蛋白，在血脑屏障（blood brain barrier, BBB）的星形胶质细胞足突高度表达，是星形胶质细胞上最为主要的功能蛋白，与水分子的转运密切相关。AQP4的调控影响着BBB的通透性变化，同样在缺血缺氧脑水肿的发生发展进程中扮演重要角色。 课题前期动物实验部分发现，HBOP能通过下调P2X7r和AQP4蛋白的表达起到对小鼠MCAO模型的保护作用。应用P2X7r的激动剂BzATP在MCAO之前对小鼠进行预处理，能上调AQP4表达，加重实验动物的神经功能损伤。这说明P2X7r对AQP4具有调控作用。因此本课题在前期动物实验的基础上，以离体培养的大鼠脑星形胶质细胞为研究对象，探讨HBOP对星形胶质细胞（oxygen glucose deprivation）OGD模型的缺氧保护作用，并着重研究P2X7r对AQP4的调控及其在HBOP抗细胞缺氧损伤机制中的重要地位。 方法： 1.选择出生3天以内的新生大鼠，从大脑中分离星形胶质细胞，通过对传统培养技术的改进，培养出纯度高且能稳定传代的星形胶质细胞，进行免疫荧光染色鉴定。 2.传代后的细胞分为4个组：对照组（control）、高压氧预适应组（HBOP）、单纯缺氧组（OGD）、高压氧预适应缺氧组（HBOP+OGD）。按既定方案进行细胞处理后，分别检测各组细胞培养基、细胞裂解液的化学发光度值，细胞凋亡率，并运用WesternBlot和QT-PCR技术检测各组细胞在预定时相点P2X7r及AQP4的蛋白及mRNA表达情况。 3.细胞分为8个组： HBOP组、 HBOP+BBG组、 HBOP+BzATP组、HBOP+BBG+BzATP组、HBOP+OGD组、HBOP+BBG+OGD组、HBOP+BzATP+OGD组、HBOP+BBG+BzATP+OGD组（BzATP和BBG分别为P2X7r的激动剂与拮抗剂）。按既定方案进行细胞处理后，进行细胞凋亡率检测，并运用Western Blot和QT-PCR技术检测各组细胞在预定时相点P2X7r及AQP4的蛋白及mRNA表达情况。 结果： 1.培养出的星形胶质细胞高度表达GFAP抗体，细胞计数分析显示纯化度高达95%； 2.HBOP具有明确的细胞缺氧保护作用；应用P2X7r的激动剂BzATP可削弱这种保护力度，加重缺氧损伤，BBG可部分拮抗BzATP的负效应； 3.HBOP可上调离体培养大鼠脑星形胶质细胞的P2X7r表达，降低AQP4蛋白表达水平；在进行OGD处理后，OGD组细胞的P2X7r和AQP4蛋白表达在缺氧6h即达到高峰，此后变化不大；而HBOP+OGD组细胞的P2X7r和AQP4蛋白表达在缺氧6h下调，此后升高，在缺氧12h时达到高峰，总体呈先降后升的趋势。QT-PCR结果与Western blot结果基本一致； 4.在正常条件下，应用BzATP后，P2X7r表达水平增加，AQP4表达减弱，应用BBG后，P2X7r表达下调，而AQP4的表达增加；在缺氧环境中，应用BzATP能同时上调P2X7r和AQP4的表达，应用BBG能同时下调P2X7r和AQP4的表达；BzATP的激动剂作用能被BBG部分拮抗。 结论： 1.HBOP处理能有效降低OGD细胞的凋亡率，具有明确的缺氧保护作用； 2.HBOP通过延迟OGD细胞的P2X7r和AQP4蛋白表达高峰起到细胞缺氧保护作用； 3.BzATP能拮抗HBOP对细胞的缺氧保护作用，而这种拮抗作用能被BBG部分抵消； 4.P2X7r对AQP4的表达具有明确的调节作用，这种调控作用在HBOP抗细胞缺氧损伤机制中占重要地位。
[Abstract]:Background and Purpose The cerebral ischemic diseases account for more than 90% of the total cerebrovascular diseases, and how to improve the tolerance of the brain tissue to the hypoxia and to reduce the nerve and functional damage after the ischemia has been a difficult and heavy research in the field of neurosurgery The results of this study have shown that the high-pressure pre-adaptation (HBOP) has a positive protective effect on spinal cord injury and cerebral ischemia, but its specific mechanism is unknown. The P2X7r (P2X7rector) is a unique subtype of the ATP-gated ion channel family, which is widely distributed in the brain tissue cells and the P2X7r is used as the upstream regulatory site, and plays a role in various functional activities in the nervous system and plays a role in the ischemic brain injury. AQP4 is a membrane water channel protein, which is highly expressed in the astrocyte of blood-brain barrier (BBB), is the most important functional protein on astrocytes, and is closely related to the transport of water molecules. The regulation of AQP4 affects the permeability of BBB and plays a role in the development of cerebral ischemia and hypoxia. It was found that the HBOP can play a role in the MCAO model of the mouse by down-regulating the expression of P2X7r and AQP4 protein. The protective effect of P2X7r was used to pre-treat the mice before MCAO, and the expression of AQP4 could be up-regulated and the God of the experimental animals could be increased. Functional damage. This indicates that the P2X7r has an AQP4 The effect of HBOP on the OGD model of astrocyte was discussed on the basis of the animal experiment in the early stage. The role of P2X7r in the regulation of AQP4 and its mechanism of anti-cell hypoxia in HBOP important The method comprises the following steps of:1, selecting a newborn rat born within 3 days, separating the astrocyte from the brain, culturing the astrocyte with high purity and stable passage through the improvement of the traditional culture technology, 2. The cells after passage were divided into 4 groups: control group (control), hyperbaric oxygen preconditioning group (HBOP), simple hypoxia group (OGD), and high-pressure oxygen pre-adapted to hypoxia. In the group (HBOP + OGD), after the cell treatment was carried out according to the established protocol, the chemiluminescence value and the cell apoptosis rate of each group of cell culture medium and the cell lysate were respectively detected, and the P2X7r and AQP4 of each group were detected at the predetermined time by using the Western Blot and the QT-PCR technique. 3. The cell is divided into 8 groups: the HBOP stack, the HBOP + BBG group, the HBOP + BzATP group, the HBOP + BBG + BzATP group, the HBOP + OGD group, the HBOP + BBG + OGD group, the HBOP + BzATP + OGD group, the HBOP + BBG + BzATP + OGD group (BzATP and the BBG are P, respectively). 2X7r (agonist and antagonist). After the cell treatment was performed according to the established protocol, the cell apoptosis rate was detected, and the P2X7r and AQP were detected by Western Blot and QT-PCR. 4 eggs Expression of white and mRNA. Results:1. The highly expressed GFAP antibody was expressed in the cultured astrocytes. 2. The cell count analysis showed that the purification was as high as 95%;2. The HBOP had a clear cell-hypoxia protective effect; the PP2X7r agonist, Bz, was used. ATP can weaken such protection and aggravate the loss of oxygen The negative effect of BzATP can be partially antagonized by BBG.3. The expression of P2X7r in the rat brain astrocyte cultured by the HBOP can be reduced, and the expression level of AQP4 is decreased; after the OGD treatment, the P2X7r and AQP4 of the OGD group cells The expression of P75r and AQP4 in the HBOP + OGD group was down-regulated after 6 h of hypoxia, and then increased after 6 h of hypoxia. The peak was reached at 12 h of hypoxia, and the overall trend was the first step down. The QT-PC The results showed that the expression of P2X7r decreased and the expression of AQP4 decreased after the application of BzATP, and the expression of AQP4 was decreased, and the expression of AQP4 was increased. BzATP can increase the expression of P2X7r and AQP4 at the same time, and the application of BBG can lower the P2X7r and AQP4 at the same time. Expression; The agonist action of BzATP can be antagonized by the BBG part. Conclusion:1. HBOP the apoptosis rate of the OGD cells can be effectively reduced, and the HBOP can effectively reduce the apoptosis rate of the OGD cells, and the HBOP can effectively reduce the apoptosis rate of the OGD cells, The expression of P2X7r and AQP4 protein in the cells can play a role in the cell hypoxia. 3. Protective effect;3. BzATP can antagonize The anti-hypoxia protective effect of the HBOP on the cells can be partially offset by the BBG, and the expression of P2X7r on AQP4 is clear.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
本文编号：2504551
[Abstract]:Background and Purpose The cerebral ischemic diseases account for more than 90% of the total cerebrovascular diseases, and how to improve the tolerance of the brain tissue to the hypoxia and to reduce the nerve and functional damage after the ischemia has been a difficult and heavy research in the field of neurosurgery The results of this study have shown that the high-pressure pre-adaptation (HBOP) has a positive protective effect on spinal cord injury and cerebral ischemia, but its specific mechanism is unknown. The P2X7r (P2X7rector) is a unique subtype of the ATP-gated ion channel family, which is widely distributed in the brain tissue cells and the P2X7r is used as the upstream regulatory site, and plays a role in various functional activities in the nervous system and plays a role in the ischemic brain injury. AQP4 is a membrane water channel protein, which is highly expressed in the astrocyte of blood-brain barrier (BBB), is the most important functional protein on astrocytes, and is closely related to the transport of water molecules. The regulation of AQP4 affects the permeability of BBB and plays a role in the development of cerebral ischemia and hypoxia. It was found that the HBOP can play a role in the MCAO model of the mouse by down-regulating the expression of P2X7r and AQP4 protein. The protective effect of P2X7r was used to pre-treat the mice before MCAO, and the expression of AQP4 could be up-regulated and the God of the experimental animals could be increased. Functional damage. This indicates that the P2X7r has an AQP4 The effect of HBOP on the OGD model of astrocyte was discussed on the basis of the animal experiment in the early stage. The role of P2X7r in the regulation of AQP4 and its mechanism of anti-cell hypoxia in HBOP important The method comprises the following steps of:1, selecting a newborn rat born within 3 days, separating the astrocyte from the brain, culturing the astrocyte with high purity and stable passage through the improvement of the traditional culture technology, 2. The cells after passage were divided into 4 groups: control group (control), hyperbaric oxygen preconditioning group (HBOP), simple hypoxia group (OGD), and high-pressure oxygen pre-adapted to hypoxia. In the group (HBOP + OGD), after the cell treatment was carried out according to the established protocol, the chemiluminescence value and the cell apoptosis rate of each group of cell culture medium and the cell lysate were respectively detected, and the P2X7r and AQP4 of each group were detected at the predetermined time by using the Western Blot and the QT-PCR technique. 3. The cell is divided into 8 groups: the HBOP stack, the HBOP + BBG group, the HBOP + BzATP group, the HBOP + BBG + BzATP group, the HBOP + OGD group, the HBOP + BBG + OGD group, the HBOP + BzATP + OGD group, the HBOP + BBG + BzATP + OGD group (BzATP and the BBG are P, respectively). 2X7r (agonist and antagonist). After the cell treatment was performed according to the established protocol, the cell apoptosis rate was detected, and the P2X7r and AQP were detected by Western Blot and QT-PCR. 4 eggs Expression of white and mRNA. Results:1. The highly expressed GFAP antibody was expressed in the cultured astrocytes. 2. The cell count analysis showed that the purification was as high as 95%;2. The HBOP had a clear cell-hypoxia protective effect; the PP2X7r agonist, Bz, was used. ATP can weaken such protection and aggravate the loss of oxygen The negative effect of BzATP can be partially antagonized by BBG.3. The expression of P2X7r in the rat brain astrocyte cultured by the HBOP can be reduced, and the expression level of AQP4 is decreased; after the OGD treatment, the P2X7r and AQP4 of the OGD group cells The expression of P75r and AQP4 in the HBOP + OGD group was down-regulated after 6 h of hypoxia, and then increased after 6 h of hypoxia. The peak was reached at 12 h of hypoxia, and the overall trend was the first step down. The QT-PC The results showed that the expression of P2X7r decreased and the expression of AQP4 decreased after the application of BzATP, and the expression of AQP4 was decreased, and the expression of AQP4 was increased. BzATP can increase the expression of P2X7r and AQP4 at the same time, and the application of BBG can lower the P2X7r and AQP4 at the same time. Expression; The agonist action of BzATP can be antagonized by the BBG part. Conclusion:1. HBOP the apoptosis rate of the OGD cells can be effectively reduced, and the HBOP can effectively reduce the apoptosis rate of the OGD cells, and the HBOP can effectively reduce the apoptosis rate of the OGD cells, The expression of P2X7r and AQP4 protein in the cells can play a role in the cell hypoxia. 3. Protective effect;3. BzATP can antagonize The anti-hypoxia protective effect of the HBOP on the cells can be partially offset by the BBG, and the expression of P2X7r on AQP4 is clear.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741
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