P2X7r調(diào)控AQP4表達(dá)在HBOP機(jī)制中的作用
發(fā)布時(shí)間：2019-06-22 11:49
【摘要】：背景和目的 腦缺血性疾病占腦血管疾病總數(shù)的90%以上，如何提高腦組織對(duì)缺氧的耐受，減輕缺血后神經(jīng)及功能損傷，，一直是神經(jīng)外科領(lǐng)域研究的難點(diǎn)和重點(diǎn)。本課題前期試驗(yàn)發(fā)現(xiàn)，高壓氧預(yù)適應(yīng)(hyperbaric oxygen preconditioning，HBOP)對(duì)脊髓損傷、腦缺血都表現(xiàn)出了積極的保護(hù)作用，但其具體作用機(jī)制尚不明確。 P2X7r（P2X7receptor）是ATP門控離子通道家族中的獨(dú)特亞型，廣泛分布于腦組織細(xì)胞，P2X7r作為上游調(diào)節(jié)位點(diǎn)，在神經(jīng)系統(tǒng)參與多種功能活動(dòng)，在缺血性腦損傷中發(fā)揮重要作用。AQP4是膜水通道蛋白，在血腦屏障（blood brain barrier, BBB）的星形膠質(zhì)細(xì)胞足突高度表達(dá)，是星形膠質(zhì)細(xì)胞上最為主要的功能蛋白，與水分子的轉(zhuǎn)運(yùn)密切相關(guān)。AQP4的調(diào)控影響著BBB的通透性變化，同樣在缺血缺氧腦水腫的發(fā)生發(fā)展進(jìn)程中扮演重要角色。 課題前期動(dòng)物實(shí)驗(yàn)部分發(fā)現(xiàn)，HBOP能通過下調(diào)P2X7r和AQP4蛋白的表達(dá)起到對(duì)小鼠MCAO模型的保護(hù)作用。應(yīng)用P2X7r的激動(dòng)劑BzATP在MCAO之前對(duì)小鼠進(jìn)行預(yù)處理，能上調(diào)AQP4表達(dá)，加重實(shí)驗(yàn)動(dòng)物的神經(jīng)功能損傷。這說明P2X7r對(duì)AQP4具有調(diào)控作用。因此本課題在前期動(dòng)物實(shí)驗(yàn)的基礎(chǔ)上，以離體培養(yǎng)的大鼠腦星形膠質(zhì)細(xì)胞為研究對(duì)象，探討HBOP對(duì)星形膠質(zhì)細(xì)胞（oxygen glucose deprivation）OGD模型的缺氧保護(hù)作用，并著重研究P2X7r對(duì)AQP4的調(diào)控及其在HBOP抗細(xì)胞缺氧損傷機(jī)制中的重要地位。 方法： 1.選擇出生3天以內(nèi)的新生大鼠，從大腦中分離星形膠質(zhì)細(xì)胞，通過對(duì)傳統(tǒng)培養(yǎng)技術(shù)的改進(jìn)，培養(yǎng)出純度高且能穩(wěn)定傳代的星形膠質(zhì)細(xì)胞，進(jìn)行免疫熒光染色鑒定。 2.傳代后的細(xì)胞分為4個(gè)組：對(duì)照組（control）、高壓氧預(yù)適應(yīng)組（HBOP）、單純?nèi)毖踅M（OGD）、高壓氧預(yù)適應(yīng)缺氧組（HBOP+OGD）。按既定方案進(jìn)行細(xì)胞處理后，分別檢測(cè)各組細(xì)胞培養(yǎng)基、細(xì)胞裂解液的化學(xué)發(fā)光度值，細(xì)胞凋亡率，并運(yùn)用WesternBlot和QT-PCR技術(shù)檢測(cè)各組細(xì)胞在預(yù)定時(shí)相點(diǎn)P2X7r及AQP4的蛋白及mRNA表達(dá)情況。 3.細(xì)胞分為8個(gè)組： HBOP組、 HBOP+BBG組、 HBOP+BzATP組、HBOP+BBG+BzATP組、HBOP+OGD組、HBOP+BBG+OGD組、HBOP+BzATP+OGD組、HBOP+BBG+BzATP+OGD組（BzATP和BBG分別為P2X7r的激動(dòng)劑與拮抗劑）。按既定方案進(jìn)行細(xì)胞處理后，進(jìn)行細(xì)胞凋亡率檢測(cè)，并運(yùn)用Western Blot和QT-PCR技術(shù)檢測(cè)各組細(xì)胞在預(yù)定時(shí)相點(diǎn)P2X7r及AQP4的蛋白及mRNA表達(dá)情況。 結(jié)果： 1.培養(yǎng)出的星形膠質(zhì)細(xì)胞高度表達(dá)GFAP抗體，細(xì)胞計(jì)數(shù)分析顯示純化度高達(dá)95%； 2.HBOP具有明確的細(xì)胞缺氧保護(hù)作用；應(yīng)用P2X7r的激動(dòng)劑BzATP可削弱這種保護(hù)力度，加重缺氧損傷，BBG可部分拮抗BzATP的負(fù)效應(yīng)； 3.HBOP可上調(diào)離體培養(yǎng)大鼠腦星形膠質(zhì)細(xì)胞的P2X7r表達(dá)，降低AQP4蛋白表達(dá)水平；在進(jìn)行OGD處理后，OGD組細(xì)胞的P2X7r和AQP4蛋白表達(dá)在缺氧6h即達(dá)到高峰，此后變化不大；而HBOP+OGD組細(xì)胞的P2X7r和AQP4蛋白表達(dá)在缺氧6h下調(diào)，此后升高，在缺氧12h時(shí)達(dá)到高峰，總體呈先降后升的趨勢(shì)。QT-PCR結(jié)果與Western blot結(jié)果基本一致； 4.在正常條件下，應(yīng)用BzATP后，P2X7r表達(dá)水平增加，AQP4表達(dá)減弱，應(yīng)用BBG后，P2X7r表達(dá)下調(diào)，而AQP4的表達(dá)增加；在缺氧環(huán)境中，應(yīng)用BzATP能同時(shí)上調(diào)P2X7r和AQP4的表達(dá)，應(yīng)用BBG能同時(shí)下調(diào)P2X7r和AQP4的表達(dá)；BzATP的激動(dòng)劑作用能被BBG部分拮抗。 結(jié)論： 1.HBOP處理能有效降低OGD細(xì)胞的凋亡率，具有明確的缺氧保護(hù)作用； 2.HBOP通過延遲OGD細(xì)胞的P2X7r和AQP4蛋白表達(dá)高峰起到細(xì)胞缺氧保護(hù)作用； 3.BzATP能拮抗HBOP對(duì)細(xì)胞的缺氧保護(hù)作用，而這種拮抗作用能被BBG部分抵消； 4.P2X7r對(duì)AQP4的表達(dá)具有明確的調(diào)節(jié)作用，這種調(diào)控作用在HBOP抗細(xì)胞缺氧損傷機(jī)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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741
本文編號(hào)：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.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741
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