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  本文選題：P2X7受體 + ATP��； 參考：《第二軍醫(yī)大學》2007年碩士論文

【摘要】： 嘌呤受體分為P1和P2兩大類。P1受體主要由腺苷(A)激活,P2受體主要由核苷酸激活,包括三磷酸腺苷(ATP)。P2受體根據(jù)其作用機理的不同又可以分為P2X和P2Y受體。P2Y受體家族(P2Y1、P2Y2、P2Y4、P2Y11-13)是一類代謝型的與G-蛋白相偶聯(lián)的受體,與磷脂酶C的激活和三磷酸肌醇的形成有關(guān)。P2X受體家族是一類對鉀、鈉和鈣可通透的離子型配體門控通道。目前已經(jīng)克隆出7個不同的P2X亞型,分別稱為P2X1-7,P2X7又稱P2Z。P2X7受體特性與其他六個亞型不同:體外電生理實驗表明ATP短暫刺激該受體即可引起非選擇性小陽離子瞬時通透;如果ATP反復或持續(xù)刺激該受體即可引起非選擇性質(zhì)膜孔道開放,最終導致細胞凋亡。P2受體廣泛分布于機體各組織器官,目前認為小膠質(zhì)細胞上表達有多種嘌呤受體亞型,包括P2X7、P2Y2和P2Y12。這表明ATP可能對小膠質(zhì)細胞有重要的調(diào)節(jié)作用。細胞外ATP可以通過P2X受體激活膠質(zhì)細胞。在體內(nèi),細胞外ATP濃度很低,不足以激活膠質(zhì)細胞,表達更多的P2X受體;而細胞內(nèi)ATP濃度較高,后者是前者的1000倍,達毫摩爾水平。在某些病理條件下如腦外傷、缺血、炎癥等局部細胞受到損傷而釋放出大量ATP及降解產(chǎn)物,這些物質(zhì)在局部濃度增高,并逐漸向鄰近區(qū)域擴散,較長時間刺激損傷區(qū)及其鄰近區(qū)的膠質(zhì)細胞。這足以激活膠質(zhì)細胞,并使P2X受體表達上調(diào)。小膠質(zhì)細胞激活后肥大、增生,形成典型的吞噬細胞,可產(chǎn)生許多生物活性物質(zhì)如活性氧、一氧化氮、蛋白酶及一些炎性物質(zhì)對周圍細胞產(chǎn)生損害作用;但另一方面又產(chǎn)生轉(zhuǎn)化因子beta 1和神經(jīng)生長因子等物質(zhì)促進神經(jīng)組織再生及修復�；罨男∧z質(zhì)細胞在腦外傷、缺血、炎癥、AIDS、多發(fā)性硬化病、蛋白病毒病、退行性病變?nèi)鏏lzheimer及Parkinson病以及衰老等過程中有著很重要的作用。 盡管上述病理過程均與小膠質(zhì)細胞活化有關(guān),但小膠質(zhì)細胞被激活的機制還不太清楚。綜合本研究和以往文獻資料,P2X7參與小膠質(zhì)細胞的激活和凋亡效應。如果用特異性拮抗劑阻斷ATP的作用,將阻止小膠質(zhì)細胞和星型膠質(zhì)細胞激活后所帶來的對周圍組織細胞的損害作用和膠質(zhì)疤痕的形成,從而保護腦組織不受進一步的損害或有利于神經(jīng)纖維的再生。 為了研究P2X7受體介導的小膠質(zhì)細胞的形態(tài)變化和受體表達情況,我們利用原代培養(yǎng)的新生大鼠小膠質(zhì)細胞進行實驗。取新生1-2 d SD大鼠的腦組織(去腦膜),含10%胎牛血清的DMEM培養(yǎng)基培養(yǎng)10-15 d,恒溫搖床輕柔轉(zhuǎn)動15 min取懸浮細胞,十二孔板鋪片20 min換液培養(yǎng)過夜。分別用0 mmol/L、1 mmol/L、2 mmol/L、3 mmol/L濃度的ATP刺激小膠質(zhì)細胞1 h,福爾馬林固定后加入第一抗體1:500稀釋和ED-1抗體,濕盒中暗處室溫放置過夜,加入稀釋的Cyc3標記的驢抗兔IgG 1:5000稀釋,室溫放置1 h。Hoechst核染色2 min,熒光顯微鏡下觀察。ED-1標記的細胞占所有細胞99%以上,表明我們原代培養(yǎng)的用于實驗的小膠質(zhì)細胞純度很高,可以滿足實驗要求。細胞在不同濃度ATP的刺激下,其形態(tài)逐漸發(fā)生了變化,細胞的偽足逐漸消失,細胞逐漸收縮變圓,P2X7受體的表達量也隨著ATP濃度的增加而增加。該實驗結(jié)果表明在P2X7受體的介導下,原代培養(yǎng)的小膠質(zhì)細胞在ATP刺激下,細胞形態(tài)逐漸發(fā)生變化,受體表達也逐漸增加,持續(xù)的ATP刺激還會導致小膠質(zhì)細胞的凋亡。 在細胞外ATP的作用下,P2X7受體可能參與了小膠質(zhì)細胞的活化和凋亡的過程。為了進一步驗證ATP對P2X7受體的作用,我們用P2X7受體的激動劑ATP和特異性拮抗劑oATP作用于原代小膠質(zhì)細胞,通過流式細胞技術(shù)來確定P2X7受體是否在小膠質(zhì)細胞凋亡中起作用。兩組原代小膠質(zhì)細胞分別加入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雙標染色,流式細胞儀檢測凋亡細胞百分率。統(tǒng)計檢驗分析顯示,ATP組和拮抗劑組的凋亡和死亡百分率存在顯著差異,這表明P2X7受體確實參與了小膠質(zhì)細胞的激活和凋亡過程。 由于原代培養(yǎng)的小膠質(zhì)細胞表達多種嘌呤受體,除了P2X7受體外,還有P2Y2和P2Y12等受體,為了排除其他受體對P2X7受體介導的小膠質(zhì)細胞形態(tài)變化和受體表達的影響,我們首次使用大鼠P2X7受體基因(GB號X95882)與EGFP基因融合構(gòu)建了真核表達重組質(zhì)粒pcDNA3-hi-EGFP-rP2X7。在此基礎上,轉(zhuǎn)染人HEK293細胞,使用G418作為篩選劑,在0.8 mg/ml濃度下,經(jīng)過單克隆化,建立了穩(wěn)定表達P2X7受體和EGFP的HEK293細胞的細胞株,經(jīng)胞外ATP刺激的功能分析和免疫組織化學等測試,該細胞株正確表達P2X7受體并穩(wěn)定傳代50代以上。 根據(jù)文獻資料可以知道,以往的實驗沒有用ATP刺激單獨表達P2X7受體的單克隆細胞模型來分析細胞的形態(tài)變化和受體表達情況。本研究利用實驗室構(gòu)建的單獨表達P2X7受體的人HEK293細胞株,分別用0 mmol/L、0.05 mmol/L、0.1 mmol/L、0.4mmol/L和0.8 mmol/L濃度的ATP去刺激在十二孔板中過夜培養(yǎng)在蓋玻片上的HEK293細胞,處理時間為1 h,福爾馬林固定后第一抗體1:500稀釋,濕盒中暗處室溫放置過夜,Cyc3標記的驢抗兔第二抗體1:5000稀釋后,濕盒中暗處室溫放置1 h,封片顯微鏡下觀察。經(jīng)ATP刺激的表達P2X7受體的HEK293細胞的形態(tài)發(fā)生明顯的變化,從低濃度ATP刺激組到高濃度ATP刺激組原本多邊形的細胞形態(tài)逐漸變圓,在細胞膜上表達的P2X7受體也隨著濃度的增加其表達量也逐漸增多。這表明ATP確實是P2X7受體的激動劑,并且在不同濃度的ATP作用下,通過單一的P2X7受體可以引起細胞的形態(tài)變化和受體表達的變化。在ATP持續(xù)刺激下,會導致細胞的凋亡。而且由于是ATP刺激單獨表達P2X7受體的單克隆細胞模型,這也在一定程度上排除了其他嘌呤受體對ATP刺激后產(chǎn)生反應的干擾。 本研究通過ATP刺激原代培養(yǎng)的小膠質(zhì)細胞,研究P2X7受體介導的細胞形態(tài)變化和受體表達情況,并且通過流式細胞技術(shù)來研究P2X7受體介導的ATP對小膠質(zhì)細胞凋亡的影響。為了排除其他嘌呤受體對P2X7受體的影響,我們首次通過建立單獨表達P2X7受體的單克隆細胞模型,對ATP作用下P2X7受體介導細胞形態(tài)變化和受體的表達進行了分析,確認了P2X7受體介導ATP激活和誘導小膠質(zhì)細胞的作用,有助于進一步研究P2X7受體信號通路及其下游過程。
[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.
【學位授予單位】：第二軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2007
【分類號】：R341

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