P2X7受体介导的小胶质细胞形态变化和受体表达的研究

发布时间：2018-07-05 21:02

本文选题：P2X7受体 + ATP　；参考：《第二军医大学》2007年硕士论文

【摘要】： 嘌呤受体分为P1和P2两大类。P1受体主要由腺苷(A)激活,P2受体主要由核苷酸激活,包括三磷酸腺苷(ATP)。P2受体根据其作用机理的不同又可以分为P2X和P2Y受体。P2Y受体家族(P2Y1、P2Y2、P2Y4、P2Y11-13)是一类代谢型的与G-蛋白相偶联的受体,与磷脂酶C的激活和三磷酸肌醇的形成有关。P2X受体家族是一类对钾、钠和钙可通透的离子型配体门控通道。目前已经克隆出7个不同的P2X亚型,分别称为P2X1-7,P2X7又称P2Z。P2X7受体特性与其他六个亚型不同:体外电生理实验表明ATP短暂刺激该受体即可引起非选择性小阳离子瞬时通透;如果ATP反复或持续刺激该受体即可引起非选择性质膜孔道开放,最终导致细胞凋亡。P2受体广泛分布于机体各组织器官,目前认为小胶质细胞上表达有多种嘌呤受体亚型,包括P2X7、P2Y2和P2Y12。这表明ATP可能对小胶质细胞有重要的调节作用。细胞外ATP可以通过P2X受体激活胶质细胞。在体内,细胞外ATP浓度很低,不足以激活胶质细胞,表达更多的P2X受体;而细胞内ATP浓度较高,后者是前者的1000倍,达毫摩尔水平。在某些病理条件下如脑外伤、缺血、炎症等局部细胞受到损伤而释放出大量ATP及降解产物,这些物质在局部浓度增高,并逐渐向邻近区域扩散,较长时间刺激损伤区及其邻近区的胶质细胞。这足以激活胶质细胞,并使P2X受体表达上调。小胶质细胞激活后肥大、增生,形成典型的吞噬细胞,可产生许多生物活性物质如活性氧、一氧化氮、蛋白酶及一些炎性物质对周围细胞产生损害作用;但另一方面又产生转化因子beta 1和神经生长因子等物质促进神经组织再生及修复。活化的小胶质细胞在脑外伤、缺血、炎症、AIDS、多发性硬化病、蛋白病毒病、退行性病变如Alzheimer及Parkinson病以及衰老等过程中有着很重要的作用。尽管上述病理过程均与小胶质细胞活化有关,但小胶质细胞被激活的机制还不太清楚。综合本研究和以往文献资料,P2X7参与小胶质细胞的激活和凋亡效应。如果用特异性拮抗剂阻断ATP的作用,将阻止小胶质细胞和星型胶质细胞激活后所带来的对周围组织细胞的损害作用和胶质疤痕的形成,从而保护脑组织不受进一步的损害或有利于神经纤维的再生。为了研究P2X7受体介导的小胶质细胞的形态变化和受体表达情况,我们利用原代培养的新生大鼠小胶质细胞进行实验。取新生1-2 d SD大鼠的脑组织(去脑膜),含10%胎牛血清的DMEM培养基培养10-15 d,恒温摇床轻柔转动15 min取悬浮细胞,十二孔板铺片20 min换液培养过夜。分别用0 mmol/L、1 mmol/L、2 mmol/L、3 mmol/L浓度的ATP刺激小胶质细胞1 h,福尔马林固定后加入第一抗体1:500稀释和ED-1抗体,湿盒中暗处室温放置过夜,加入稀释的Cyc3标记的驴抗兔IgG 1:5000稀释,室温放置1 h。Hoechst核染色2 min,荧光显微镜下观察。ED-1标记的细胞占所有细胞99%以上,表明我们原代培养的用于实验的小胶质细胞纯度很高,可以满足实验要求。细胞在不同浓度ATP的刺激下,其形态逐渐发生了变化,细胞的伪足逐渐消失,细胞逐渐收缩变圆,P2X7受体的表达量也随着ATP浓度的增加而增加。该实验结果表明在P2X7受体的介导下,原代培养的小胶质细胞在ATP刺激下,细胞形态逐渐发生变化,受体表达也逐渐增加,持续的ATP刺激还会导致小胶质细胞的凋亡。在细胞外ATP的作用下,P2X7受体可能参与了小胶质细胞的活化和凋亡的过程。为了进一步验证ATP对P2X7受体的作用,我们用P2X7受体的激动剂ATP和特异性拮抗剂oATP作用于原代小胶质细胞,通过流式细胞技术来确定P2X7受体是否在小胶质细胞凋亡中起作用。两组原代小胶质细胞分别加入3 mmol/L ATP和3 mmol/L ATP+0.1 mmol/L oATP作用15 min、30min、60min、90 min和120 min,Annexin V/PI双标染色,流式细胞仪检测凋亡细胞百分率。统计检验分析显示,ATP组和拮抗剂组的凋亡和死亡百分率存在显著差异,这表明P2X7受体确实参与了小胶质细胞的激活和凋亡过程。由于原代培养的小胶质细胞表达多种嘌呤受体,除了P2X7受体外,还有P2Y2和P2Y12等受体,为了排除其他受体对P2X7受体介导的小胶质细胞形态变化和受体表达的影响,我们首次使用大鼠P2X7受体基因(GB号X95882)与EGFP基因融合构建了真核表达重组质粒pcDNA3-hi-EGFP-rP2X7。在此基础上,转染人HEK293细胞,使用G418作为筛选剂,在0.8 mg/ml浓度下,经过单克隆化,建立了稳定表达P2X7受体和EGFP的HEK293细胞的细胞株,经胞外ATP刺激的功能分析和免疫组织化学等测试,该细胞株正确表达P2X7受体并稳定传代50代以上。根据文献资料可以知道,以往的实验没有用ATP刺激单独表达P2X7受体的单克隆细胞模型来分析细胞的形态变化和受体表达情况。本研究利用实验室构建的单独表达P2X7受体的人HEK293细胞株,分别用0 mmol/L、0.05 mmol/L、0.1 mmol/L、0.4mmol/L和0.8 mmol/L浓度的ATP去刺激在十二孔板中过夜培养在盖玻片上的HEK293细胞,处理时间为1 h,福尔马林固定后第一抗体1:500稀释,湿盒中暗处室温放置过夜,Cyc3标记的驴抗兔第二抗体1:5000稀释后,湿盒中暗处室温放置1 h,封片显微镜下观察。经ATP刺激的表达P2X7受体的HEK293细胞的形态发生明显的变化,从低浓度ATP刺激组到高浓度ATP刺激组原本多边形的细胞形态逐渐变圆,在细胞膜上表达的P2X7受体也随着浓度的增加其表达量也逐渐增多。这表明ATP确实是P2X7受体的激动剂,并且在不同浓度的ATP作用下,通过单一的P2X7受体可以引起细胞的形态变化和受体表达的变化。在ATP持续刺激下,会导致细胞的凋亡。而且由于是ATP刺激单独表达P2X7受体的单克隆细胞模型,这也在一定程度上排除了其他嘌呤受体对ATP刺激后产生反应的干扰。本研究通过ATP刺激原代培养的小胶质细胞,研究P2X7受体介导的细胞形态变化和受体表达情况,并且通过流式细胞技术来研究P2X7受体介导的ATP对小胶质细胞凋亡的影响。为了排除其他嘌呤受体对P2X7受体的影响,我们首次通过建立单独表达P2X7受体的单克隆细胞模型,对ATP作用下P2X7受体介导细胞形态变化和受体的表达进行了分析,确认了P2X7受体介导ATP激活和诱导小胶质细胞的作用,有助于进一步研究P2X7受体信号通路及其下游过程。
[Abstract]:The.P1 receptors of P1 and P2 two are mainly activated by adenosine (A), and the P2 receptor is mainly activated by nucleotides, including the.P2 receptor of adenosine triphosphate (ATP), which can be divided into P2X and P2Y receptor.P2Y receptor family according to their different mechanisms of action. The activation of enzyme C and the formation of inositol three phosphate related.P2X receptor family are a class of ionic ligand gated channels for potassium, sodium and calcium permeable ligand. 7 different P2X subtypes have been cloned, called P2X1-7, P2X7 also called P2Z.P2X7 receptor, which is different from the other six subtypes: in vitro electrophysiological experiments show that ATP stimulates the receptor briefly. The non selective small cations can be instantaneously permeable; if ATP repeatedly or continuously stimulates the receptor, it can cause the non selective membrane opening to open and eventually lead to the widespread distribution of.P2 receptors in the tissues and organs of the body. At present, a variety of opinopterin receptor subtypes are expressed on microglia, including P2X7, P2Y2 and P2Y12., which indicates A TP may play an important role in the regulation of microglia. Extracellular ATP can activate glial cells through the P2X receptor. In vivo, the extracellular ATP concentration is low enough to activate glial cells to express more P2X receptors, and the intracellular ATP concentration is high, the latter is 1000 times the former and reaches mmol level. Under certain pathological conditions, such as the brain Injury, ischemia, inflammation and other local cells are damaged and release a large number of ATP and degradation products. These substances increase in local concentration, and gradually spread to adjacent regions. Long time stimulates the glial cells in the damaged areas and adjacent areas. This is sufficient to activate glial cells and to increase the expression of P2X receptors. Microglia is activated and hypertrophic and proliferated. The formation of typical phagocytes can produce many bioactive substances, such as reactive oxygen species, nitric oxide, protease and some inflammatory substances that damage the surrounding cells; on the other hand, the transformation factor beta 1 and nerve growth factor are produced to promote the regeneration and repair of the nerve tissue. Blood, inflammation, AIDS, multiple sclerosis, protein virus disease, degenerative diseases such as Alzheimer and Parkinson disease and aging play an important role.
Although these pathological processes are related to the activation of microglia, the mechanism of the activation of microglia is not very clear. Combined with this study and previous literature, P2X7 participates in the activation and apoptosis effect of microglia. If a specific antagonist is used to block the action of ATP, the activation of microglia and astrocytes will be blocked after activation. The damage to the surrounding tissue cells and the formation of the glial scar will protect the brain tissue from further damage or to the regeneration of the nerve fibers.
In order to study the morphological changes and receptor expression of P2X7 receptor mediated microglia, we used primary cultured neonatal rat microglia to experiment. The brain tissue (meninges) of newborn 1-2 D SD rats (DMC), DMEM medium containing 10% fetal bovine serum were cultured 10-15 D, the constant temperature rocking bed was gently rotated 15 min to take suspension cells, twelve The orifice plate was replaced by 20 min for the night. The microglia 1 h was stimulated with 0 mmol/L, 1 mmol/L, 2 mmol/L, and 3 mmol/L concentration ATP. Formalin was fixed with the first antibody 1:500 dilution and ED-1 antibody, and the wet box was placed in the dark room at room temperature for the night. The diluted Cyc3 labeled ass against rabbit IgG 1:5000 was diluted, and 1 nucleation was placed at room temperature. Color 2 min, under the fluorescence microscope, the.ED-1 labeled cells accounted for more than 99% of all cells, indicating that the purity of the microglia used for experiment in our primary culture was very high and could meet the requirements of the experiment. The morphology of cells changed gradually under the stimulation of different concentrations of ATP, and the cells gradually disappeared, the cells gradually contracted and turned round, P2X7 The expression of the receptor also increased with the increase of ATP concentration. The results showed that under the guidance of P2X7 receptor, the cell morphology of the primary cultured microglia was gradually changed, the expression of the receptor was gradually increased, and the apoptosis of microglia could be induced by the continuous ATP stimulation.
Under the action of extracellular ATP, P2X7 receptor may participate in the process of activation and apoptosis of microglia. In order to further verify the effect of ATP on P2X7 receptor, we use P2X7 receptor agonist ATP and specific antagonist oATP to act on the primary microglia, and determine whether P2X7 receptors are in microglia by flow cytometry. The two groups of primary microglia were added to 3 mmol/L ATP and 3 mmol/L ATP+0.1 mmol/L oATP respectively to act as 15 min, 30min, 60min, 90 min and 120 min, Annexin, and the percentage of apoptotic cells were detected by flow cytometry. The statistical test analysis showed that the percentage of apoptosis and death in the group and the antagonist group were significant. The difference indicates that P2X7 receptor is involved in the activation and apoptosis process of microglia.
In order to exclude the effects of other receptors on the morphologic changes and receptor expression of microglia mediated by P2X7 receptor, we first use the P2X7 receptor gene (GB No. GB X95882) and EGFP gene fusion to construct a eukaryotic expression for the expression of a variety of purinergic receptors in the primary cultured microglia, in addition to P2X7 receptor in vitro, and the effects of other receptors on the morphologic changes of microglia and the expression of the receptor mediated by P2X7 receptor. On the basis of the recombinant plasmid pcDNA3-hi-EGFP-rP2X7., human HEK293 cells were transfected and G418 was used as a screening agent. Under the concentration of 0.8 mg/ml, a cell line that stably expressed P2X7 receptor and EGFP HEK293 cells was established. The function analysis and immunohistochemistry test of extracellular ATP stimulated the cell strain to express P2X7 correctly. The receptor is stable for over 50 generations.
According to the literature, the previous experiments did not use ATP to stimulate the cell model to express the P2X7 receptor alone to analyze the cell morphological changes and the receptor expression. This study uses a laboratory constructed human HEK293 cell line that expresses P2X7 receptor alone, using 0 mmol/L, 0.05 mmol/L, 0.1 mmol/L, 0.4mmol/L, and 0.8 MMO, respectively. The l/L concentration of ATP was used to stimulate the HEK293 cells on the cover glass in the twelve orifice plate, the treatment time was 1 h, the first antibody 1:500 was diluted by formalin, the dark room at room temperature was placed overnight, and the Cyc3 labeled donkey anti rabbit second antibody 1:5000 was diluted, and the dark chamber temperature was placed 1 h in the wet box, and the seal was observed under the microscope. The morphology of the stimulated expression of P2X7 receptor HEK293 cells was obviously changed. From the low concentration ATP stimulation group to the high concentration ATP stimulation group, the cell morphology of the original polygon gradually became round, and the P2X7 receptor expressed on the cell membrane also increased with the increase of concentration. This shows that ATP is indeed an agonist of the P2X7 receptor and is in the form of a P2X7 receptor. Under the action of different concentrations of ATP, the morphological change of cells and the expression of the receptor can be induced by a single P2X7 receptor. Under the continuous stimulation of ATP, the cell apoptosis can be induced. Moreover, it is a monoclonal cell model which is stimulated by ATP to express P2X7 receptor alone. This also excludes the production of other purinergic receptors on ATP stimulation at a certain degree. The interference of the reaction.
In this study, primary cultured microglia were stimulated by ATP to study the cell morphological changes and receptor expression mediated by P2X7 receptor, and the effect of P2X7 receptor mediated ATP on the apoptosis of microglia was studied by flow cytometry. In order to exclude the effect of other purine receptors on the P2X7 receptor, we first established a single cell. The monoclonal cell model of P2X7 receptor is expressed. The morphological changes of P2X7 receptors and the expression of the receptor under the action of ATP are analyzed. The effect of P2X7 receptor on ATP activation and induction of microglia is confirmed. It is helpful to further study the P2X7 receptor signaling pathway and its downstream process.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R341

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