本文選題：血管緊張素Ⅱ1型受體 + 巨噬細(xì)胞��； 參考：《安徽醫(yī)科大學(xué)》2009年碩士論文

【摘要】： 一、研究背景 膿毒癥(sepsis)是感染所致的全身炎癥反應(yīng)綜合征(systemic inflammatoryresponse syndrome,SIRS),為嚴(yán)重創(chuàng)傷、燒傷、休克、大手術(shù)后常見(jiàn)的并發(fā)癥,進(jìn)一步發(fā)展可誘發(fā)膿毒性休克、多器官功能障礙綜合征(multiple organ dysfunctionsyndrome,MODS),甚至死亡。大量的實(shí)驗(yàn)和臨床研究均證實(shí):革蘭氏陰性桿菌的內(nèi)毒素是引起膿毒癥機(jī)體炎癥反應(yīng)的主要元兇,內(nèi)毒素引起單核巨噬細(xì)胞系統(tǒng)的激活及其釋放的內(nèi)源性介質(zhì)在膿毒癥的發(fā)生和發(fā)展過(guò)程中起著關(guān)鍵作用。 血管緊張素Ⅱ(angiotensinⅡ,AngⅡ)是腎素—血管緊張素系統(tǒng)(RAS)中最具活性的成分,也是RAS中最重要的效應(yīng)因子之一。近年來(lái)研究發(fā)現(xiàn),AngⅡ與細(xì)胞表面的血管緊張素Ⅱ1型(angiotensinⅡtype 1,AT1)受體結(jié)合后,調(diào)節(jié)細(xì)胞因子、趨化因子和黏附分子等炎癥介質(zhì)的表達(dá),參與膿毒癥及機(jī)體炎癥相關(guān)疾病的發(fā)生和發(fā)展。但有關(guān)膿毒癥時(shí)巨噬細(xì)胞AT1受體的活化以及其與炎癥介質(zhì)產(chǎn)生之間的關(guān)系,目前尚不清楚。 本實(shí)驗(yàn)采用RAW 264.7巨噬細(xì)胞為主要研究對(duì)象,觀察膿毒癥時(shí)巨噬細(xì)胞AT1受體的活化規(guī)律,并使用AT1受體拮抗劑ZD7155進(jìn)行干預(yù),觀察AT1受體在巨噬細(xì)胞促炎性細(xì)胞因子TNF-α、IL-1β、IL-6和抗炎性細(xì)胞因子IL-10以及活性氧(reactiveoxygen species,ROS)產(chǎn)生中的作用,以了解AT1受體在膿毒癥時(shí)巨噬細(xì)胞炎癥介質(zhì)產(chǎn)生中的作用和機(jī)制,以進(jìn)一步認(rèn)識(shí)膿毒癥的發(fā)生機(jī)制并為其防治提供新的思路和方法。 二、研究目的 1.探討LPS和血管緊張素Ⅱ?qū)AW264.7巨噬細(xì)胞AT1受體表達(dá)的影響。 2.研究AT1受體在RAW264.7巨噬細(xì)胞促炎性細(xì)胞因子TNF-α、IL-1β、IL-6和抗炎性細(xì)胞因子IL-10以及ROS產(chǎn)生中的作用。 三、研究?jī)?nèi)容 第一部分LPS和血管緊張素Ⅱ?qū)AW264.7巨噬細(xì)胞血管緊張素Ⅱ1型受體表達(dá)的影響 小鼠RAW264.7巨噬細(xì)胞常規(guī)培養(yǎng)后分劑量效應(yīng)、時(shí)間效應(yīng)和協(xié)同效應(yīng)三方面研究。(1)劑量效應(yīng)研究:加入不同濃度的LPS(0.01,0.1,1,10,100μg/ml)和不同濃度的AngⅡ(0.001,0.01,0.1,1,10μmol/L)分別刺激24h;(2)時(shí)間效應(yīng)研究:加入0.1μg/ml的LPS或0.01μmol/L的AngⅡ分別刺激0,1,3,6,9,12和24h;(3)協(xié)同效應(yīng)研究:先加入0.1μg/ml LPS刺激3h后,再加入0.01μmol/L的AngⅡ共同刺激6h。免疫組織化學(xué)方法檢測(cè)RAW264.7巨噬細(xì)胞AT1受體的表達(dá),使用RT-PCR的方法檢測(cè)細(xì)胞AT1受體mRNA的表達(dá),并采用Western blot法檢測(cè)細(xì)胞AT1受體蛋白的表達(dá)。 第二部分血管緊張素Ⅱ1型受體在RAW264.7巨噬細(xì)胞細(xì)胞因子和氧自由基產(chǎn)生中的作用 小鼠RAW264.7巨噬細(xì)胞常規(guī)培養(yǎng)后分為8個(gè)實(shí)驗(yàn)組:(1)空白對(duì)照組:常規(guī)培養(yǎng)9h;(2)LPS組:0.1μg/ml LPS作用9h;(3)AngⅡ組:1μmol/L AngⅡ作用6h;(4)ZD7155組:預(yù)先用38μmol/L的ZD7155作用細(xì)胞1h,再常規(guī)培養(yǎng)9h;(5)LPS+AngⅡ組:0.1μg/ml LPS作用3小時(shí)后和1μmol/L AngⅡ再共同作用6h;(6)LPS+ZD7155組:預(yù)先用38μmol/L的ZD7155作用細(xì)胞1h,再用0.1μg/ml LPS作用9h;(7)AngⅡ+ZD7155組:預(yù)先用38μmol/L的ZD7155作用細(xì)胞1h,再用1μmol/L AngⅡ作用6h;(8)LPS+AngⅡ+ZD7155組:預(yù)先用38μmol/L的ZD7155作用細(xì)胞1h,再用0.1μg/ml LPS作用3小時(shí)后和1μmol/L AngⅡ再共同作用6h。使用RT-PCR方法測(cè)定RAW264.7巨噬細(xì)胞細(xì)胞因子TNF-α、IL-1β、IL-6和IL-10mRNA的表達(dá),ELISA法測(cè)定細(xì)胞培養(yǎng)上清液中上述細(xì)胞因子的含量,并采用流式細(xì)胞技術(shù)測(cè)定細(xì)胞中ROS產(chǎn)生的情況。 四、研究結(jié)果 第一部分 用免疫組織化學(xué)方法發(fā)現(xiàn)AT1受體密集表達(dá)于RAW264.7巨噬細(xì)胞膜表面。RT-PCR和western blot結(jié)果顯示:(1)LPS誘導(dǎo)RAW264.7巨噬細(xì)胞AT1表達(dá)呈劑量依賴性,在一定范圍內(nèi)(＜0.1μg/ml),隨著LPS濃度的增加,巨噬細(xì)胞AT1受體的表達(dá)和細(xì)胞內(nèi)AT1受體mRNA的表達(dá)均逐漸增強(qiáng),LPS 0.1μg/ml孵育24h誘導(dǎo)AT1表達(dá)達(dá)到高峰,但高濃度(＞1μg/ml)LPS的刺激效應(yīng)逐漸降低。(2)LPS(0.1μg/ml)誘導(dǎo)RAW264.7巨噬細(xì)胞AT1表達(dá)呈時(shí)間依賴性,孵育1h后,巨噬細(xì)胞AT1受體的表達(dá)和細(xì)胞內(nèi)AT1受體mRNA的表達(dá)均開(kāi)始增強(qiáng),在9h達(dá)到最高峰,隨后下降。(3)AngⅡ誘導(dǎo)RAW264.7巨噬細(xì)胞AT1表達(dá)呈劑量依賴性,在一定范圍內(nèi)(＜0.01μmol/L),隨著AngⅡ濃度的增加,巨噬細(xì)胞AT1受體的表達(dá)和細(xì)胞內(nèi)AT1受體mRNA的表達(dá)均逐漸增強(qiáng),AngⅡ0.01μmol/L孵育24h誘導(dǎo)AT1表達(dá)達(dá)到高峰,但高濃度(＞0.1μmol/L)AngⅡ的刺激效應(yīng)逐漸降低。(4)AngⅡ(0.01μmol/L)誘導(dǎo)RAW264.7巨噬細(xì)胞AT1表達(dá)呈時(shí)間依賴性,孵育1h后,巨噬細(xì)胞AT1受體的表達(dá)和細(xì)胞內(nèi)AT1受體mRNA的表達(dá)均開(kāi)始增強(qiáng),在6h達(dá)到最高峰,隨后下降。(5)LPS(0.1μg/m1l和AngⅡ(0.01μmol/L)共同作用后,RAW264.7巨噬細(xì)胞AT1受體的表達(dá)和細(xì)胞內(nèi)AT1受體mRNA的表達(dá)均較空白對(duì)照組顯著增強(qiáng),但與LPS或AngⅡ的單獨(dú)作用相比,差異均無(wú)統(tǒng)計(jì)學(xué)意義。 第二部分 RT-PCR和ELISA結(jié)果顯示,0.1μg/ml的LPS和1μmol/L的AngⅡ,不論兩者單獨(dú)作用還是共同作用,RAW264.7巨噬細(xì)胞中TNF-α、IL-1β、IL-6和IL-10mRNA的表達(dá)和培養(yǎng)上清液中這些細(xì)胞因子含量均較空白對(duì)照組明顯升高,使用AT1受體拈抗劑ZD7155預(yù)處理1h能明顯抑制這種升高。流式細(xì)胞儀檢測(cè)結(jié)果顯示,和空白對(duì)照組相比,LPS和AngⅡ單獨(dú)或兩者共同作用,RAW264.7巨噬細(xì)胞ROS的產(chǎn)生均明顯增加,AT1受體拮抗劑ZD7155預(yù)處理1h能顯著抑制AngⅡ誘發(fā)的ROS產(chǎn)生。 五、研究結(jié)論 1.LPS和AngⅡ可以誘導(dǎo)RAW264.7巨噬細(xì)胞AT1受體的表達(dá),這種表達(dá)呈劑量依賴性和時(shí)間依賴性變化。 2.AT1受體介導(dǎo)了LPS和AngⅡ誘導(dǎo)RAW264.7巨噬細(xì)胞促炎性細(xì)胞因子TNF-α、IL-1β、IL-6和抗炎性細(xì)胞因子IL-10的產(chǎn)生,AngⅡ通過(guò)與AT1受體結(jié)合調(diào)節(jié)RAW264.7巨噬細(xì)胞ROS的產(chǎn)生。
[Abstract]:First, research background
Sepsis (sepsis) is a systemic inflammatory response syndrome (systemic inflammatoryresponse syndrome, SIRS) caused by infection, which is a common complication after severe trauma, burns, shock and major surgery. Further development can induce septic shock, multiple organ dysfunction syndrome (multiple organ dysfunctionsyndrome, MODS), and even death. Both experimental and clinical studies have confirmed that the endotoxin of gram-negative bacilli is the main cause of inflammatory response to sepsis, and the activation of the mononuclear macrophage system and the release of endogenous mediators in the endotoxin play a key role in the development and development of sepsis.
Angiotensin II (angiotensin II, Ang II) is the most active component in the renin angiotensin system (RAS) and is one of the most important effector factors in RAS. In recent years, studies have found that Ang II regulates cytokines, chemokines and adhesion after the binding of the angiotensin II (angiotensin type 1, AT1) receptor on the surface of the cell surface (angiotensin II type 1, AT1). The expression of inflammatory mediators, such as molecules, is involved in the development and development of sepsis and inflammation related diseases. However, the relationship between the activation of AT1 receptor in macrophages and its relationship with the production of inflammatory mediators in sepsis is not yet clear.
In this experiment, RAW 264.7 macrophages were used as the main research object to observe the activation of macrophage AT1 receptor in sepsis and the use of AT1 receptor antagonist ZD7155 to observe the AT1 receptor in macrophage proinflammatory cytokines TNF- alpha, IL-1 beta, IL-6, and anti inflammatory cell factor IL-10 and reactive oxygen species (reactiveoxygen species, ROS). To understand the role and mechanism of AT1 receptor in the production of macrophage inflammatory mediators in sepsis to further understand the mechanism of sepsis and provide new ideas and methods for the prevention and treatment of sepsis.
Two, the purpose of the study
1. to investigate the effects of LPS and angiotensin II on the expression of AT1 receptor in RAW264.7 macrophages.
2. to study the role of AT1 receptor in the proinflammatory cytokine TNF- alpha, IL-1 beta, IL-6 and anti-inflammatory cytokines IL-10 and ROS production in RAW264.7 macrophages.
Three, research content
Part one the effect of LPS and angiotensin II on the expression of angiotensin II type 1 receptor in RAW264.7 macrophages
Three aspects of dose effect, time effect and synergistic effect were studied in mice RAW264.7 macrophages. (1) dose effect study: adding different concentrations of LPS (0.01,0.1,1,10100 mu g/ml) and Ang II (0.001,0.01,0.1,1,10 mol/L) of different concentrations to stimulate 24h; (2) time effect study: 0.1 micron LPS or 0.01 micron mol/L Ang II stimulated 0,1,3,6,9,12 and 24h respectively; (3) synergistic effect study: after adding 0.1 mu g/ml LPS to stimulate 3h, and then adding 0.01 mu mol/L to stimulate 6h. immunohistochemical method to detect the expression of AT1 receptor in RAW264.7 macrophages. Expression of T1 receptor protein.
The second part is the role of angiotensin II type 1 receptor in the production of cytokines and oxygen free radicals in RAW264.7 macrophages.
The normal culture of mouse RAW264.7 macrophages was divided into 8 experimental groups: (1) blank control group: routine culture of 9h; (2) group LPS: 0.1 mu g/ml LPS action 9h; (3) Ang II Group: 1 mu mol/L Ang II action 6h; (4) ZD7155 group: 38 micron mol/L cells in advance; and (5) 0.1 mu (0.1 mu) after 3 hours and 1 mu II. (6) 6h (6) group LPS+ZD7155: ZD7155 cell 1h in advance, 9h with 0.1 micron g/ml LPS, and (7) Ang II +ZD7155 group: 38 micron mol/L ZD7155 cells in advance, and 1 micron. After the co action of 1 mu mol/L Ang II, 6h. was used to determine the expression of RAW264.7 macrophage cytokines TNF- alpha, IL-1 beta, IL-6 and IL-10mRNA, and the ELISA assay was used to determine the content of the above cytokines in the cell culture supernatant, and the flow cytometry was used to determine the ROS production in the cells.
Four, the results of the study
Part one
The results of AT1 receptor expression on the surface of RAW264.7 macrophage membrane.RT-PCR and Western blot showed that: (1) LPS induced AT1 expression in RAW264.7 macrophages was dose-dependent, within a certain range (< 0.1 g/ml). With the increase of LPS concentration, the expression of AT1 receptors in macrophages and the intracellular AT1 receptors The expression increased gradually. The expression of AT1 in 24h induced by LPS 0.1 mu g/ml reached the peak, but the stimulation effect of high concentration (> 1 mu g/ml) LPS decreased gradually. (2) LPS (0.1 mu g/ml) induced RAW264.7 macrophage AT1 expression to be time dependent. After incubating 1H, the expression of macrophage AT1 receptor and the expression of the receptor in cell began to increase. To the peak, then decreased. (3) Ang II induced RAW264.7 macrophage AT1 expression in a dose dependent manner, in a certain range (< 0.01 mol/L). With the increase of Ang II concentration, the expression of AT1 receptor in macrophages and the expression of AT1 receptor mRNA in the cells increased gradually. Ang II 0.01 micron mol/L incubated 24h inducible AT1 expression reached the peak, but high concentration ( The stimulation effect of Ang II was gradually reduced. (4) Ang II (0.01 mu mol/L) induced RAW264.7 macrophage AT1 expression to be time dependent. After incubating 1H, the expression of AT1 receptor in macrophages and the expression of AT1 receptor mRNA in the cell began to increase, and in 6h reached the peak, then decreased. (5) LPS (0.1 micron and 0.01 micron) Co acted. After use, the expression of AT1 receptor in RAW264.7 macrophages and the expression of AT1 receptor mRNA in the cells were significantly higher than that in the blank control group, but the difference was not statistically significant compared with the individual action of LPS or Ang II.
The second part
The results of RT-PCR and ELISA showed that 0.1 mu g/ml LPS and 1 mu mol/L Ang II, regardless of their individual action or common action, the content of these cytokines in the expression of TNF- alpha, IL-1 beta, IL-6 and IL-10mRNA in the RAW264.7 macrophages and the cultured supernatants were significantly higher than that in the blank control group. The flow cytometry showed that, compared with the blank control group, the production of ROS in RAW264.7 macrophages was significantly increased by both LPS and Ang II, and ZD7155 pretreated with AT1 receptor antagonist ZD7155 could significantly inhibit the production of ROS induced by Ang II.
Five, the conclusion of the study
1.LPS and Ang II can induce the expression of AT1 receptor in RAW264.7 macrophages, which is dose dependent and time-dependent.
2.AT1 receptors mediate LPS and Ang II inducing the production of TNF- alpha, IL-1 beta, IL-6, and anti-inflammatory cytokine IL-10 of RAW264.7 macrophages, and Ang II regulates RAW264.7 macrophage production by binding to AT1 receptors.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R363

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相關(guān)期刊論文 前1條

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