血管紧张素Ⅱ1型受体在RAW264.7巨噬细胞中的表达及其在炎症介质产生中的作用

发布时间：2018-06-28 02:40

本文选题：血管紧张素Ⅱ1型受体 + 巨噬细胞　；参考：《安徽医科大学》2009年硕士论文

【摘要】： 一、研究背景脓毒症(sepsis)是感染所致的全身炎症反应综合征(systemic inflammatoryresponse syndrome,SIRS),为严重创伤、烧伤、休克、大手术后常见的并发症,进一步发展可诱发脓毒性休克、多器官功能障碍综合征(multiple organ dysfunctionsyndrome,MODS),甚至死亡。大量的实验和临床研究均证实:革兰氏阴性杆菌的内毒素是引起脓毒症机体炎症反应的主要元凶,内毒素引起单核巨噬细胞系统的激活及其释放的内源性介质在脓毒症的发生和发展过程中起着关键作用。血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)是肾素—血管紧张素系统(RAS)中最具活性的成分,也是RAS中最重要的效应因子之一。近年来研究发现,AngⅡ与细胞表面的血管紧张素Ⅱ1型(angiotensinⅡtype 1,AT1)受体结合后,调节细胞因子、趋化因子和黏附分子等炎症介质的表达,参与脓毒症及机体炎症相关疾病的发生和发展。但有关脓毒症时巨噬细胞AT1受体的活化以及其与炎症介质产生之间的关系,目前尚不清楚。本实验采用RAW 264.7巨噬细胞为主要研究对象,观察脓毒症时巨噬细胞AT1受体的活化规律,并使用AT1受体拮抗剂ZD7155进行干预,观察AT1受体在巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和抗炎性细胞因子IL-10以及活性氧(reactiveoxygen species,ROS)产生中的作用,以了解AT1受体在脓毒症时巨噬细胞炎症介质产生中的作用和机制,以进一步认识脓毒症的发生机制并为其防治提供新的思路和方法。二、研究目的 1.探讨LPS和血管紧张素Ⅱ对RAW264.7巨噬细胞AT1受体表达的影响。 2.研究AT1受体在RAW264.7巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和抗炎性细胞因子IL-10以及ROS产生中的作用。三、研究内容第一部分LPS和血管紧张素Ⅱ对RAW264.7巨噬细胞血管紧张素Ⅱ1型受体表达的影响小鼠RAW264.7巨噬细胞常规培养后分剂量效应、时间效应和协同效应三方面研究。(1)剂量效应研究:加入不同浓度的LPS(0.01,0.1,1,10,100μg/ml)和不同浓度的AngⅡ(0.001,0.01,0.1,1,10μmol/L)分别刺激24h;(2)时间效应研究:加入0.1μg/ml的LPS或0.01μmol/L的AngⅡ分别刺激0,1,3,6,9,12和24h;(3)协同效应研究:先加入0.1μg/ml LPS刺激3h后,再加入0.01μmol/L的AngⅡ共同刺激6h。免疫组织化学方法检测RAW264.7巨噬细胞AT1受体的表达,使用RT-PCR的方法检测细胞AT1受体mRNA的表达,并采用Western blot法检测细胞AT1受体蛋白的表达。第二部分血管紧张素Ⅱ1型受体在RAW264.7巨噬细胞细胞因子和氧自由基产生中的作用小鼠RAW264.7巨噬细胞常规培养后分为8个实验组:(1)空白对照组:常规培养9h;(2)LPS组:0.1μg/ml LPS作用9h;(3)AngⅡ组:1μmol/L AngⅡ作用6h;(4)ZD7155组:预先用38μmol/L的ZD7155作用细胞1h,再常规培养9h;(5)LPS+AngⅡ组:0.1μg/ml LPS作用3小时后和1μmol/L AngⅡ再共同作用6h;(6)LPS+ZD7155组:预先用38μmol/L的ZD7155作用细胞1h,再用0.1μg/ml LPS作用9h;(7)AngⅡ+ZD7155组:预先用38μmol/L的ZD7155作用细胞1h,再用1μmol/L AngⅡ作用6h;(8)LPS+AngⅡ+ZD7155组:预先用38μmol/L的ZD7155作用细胞1h,再用0.1μg/ml LPS作用3小时后和1μmol/L AngⅡ再共同作用6h。使用RT-PCR方法测定RAW264.7巨噬细胞细胞因子TNF-α、IL-1β、IL-6和IL-10mRNA的表达,ELISA法测定细胞培养上清液中上述细胞因子的含量,并采用流式细胞技术测定细胞中ROS产生的情况。四、研究结果第一部分用免疫组织化学方法发现AT1受体密集表达于RAW264.7巨噬细胞膜表面。RT-PCR和western blot结果显示:(1)LPS诱导RAW264.7巨噬细胞AT1表达呈剂量依赖性,在一定范围内(＜0.1μg/ml),随着LPS浓度的增加,巨噬细胞AT1受体的表达和细胞内AT1受体mRNA的表达均逐渐增强,LPS 0.1μg/ml孵育24h诱导AT1表达达到高峰,但高浓度(＞1μg/ml)LPS的刺激效应逐渐降低。(2)LPS(0.1μg/ml)诱导RAW264.7巨噬细胞AT1表达呈时间依赖性,孵育1h后,巨噬细胞AT1受体的表达和细胞内AT1受体mRNA的表达均开始增强,在9h达到最高峰,随后下降。(3)AngⅡ诱导RAW264.7巨噬细胞AT1表达呈剂量依赖性,在一定范围内(＜0.01μmol/L),随着AngⅡ浓度的增加,巨噬细胞AT1受体的表达和细胞内AT1受体mRNA的表达均逐渐增强,AngⅡ0.01μmol/L孵育24h诱导AT1表达达到高峰,但高浓度(＞0.1μmol/L)AngⅡ的刺激效应逐渐降低。(4)AngⅡ(0.01μmol/L)诱导RAW264.7巨噬细胞AT1表达呈时间依赖性,孵育1h后,巨噬细胞AT1受体的表达和细胞内AT1受体mRNA的表达均开始增强,在6h达到最高峰,随后下降。(5)LPS(0.1μg/m1l和AngⅡ(0.01μmol/L)共同作用后,RAW264.7巨噬细胞AT1受体的表达和细胞内AT1受体mRNA的表达均较空白对照组显著增强,但与LPS或AngⅡ的单独作用相比,差异均无统计学意义。第二部分 RT-PCR和ELISA结果显示,0.1μg/ml的LPS和1μmol/L的AngⅡ,不论两者单独作用还是共同作用,RAW264.7巨噬细胞中TNF-α、IL-1β、IL-6和IL-10mRNA的表达和培养上清液中这些细胞因子含量均较空白对照组明显升高,使用AT1受体拈抗剂ZD7155预处理1h能明显抑制这种升高。流式细胞仪检测结果显示,和空白对照组相比,LPS和AngⅡ单独或两者共同作用,RAW264.7巨噬细胞ROS的产生均明显增加,AT1受体拮抗剂ZD7155预处理1h能显著抑制AngⅡ诱发的ROS产生。五、研究结论 1.LPS和AngⅡ可以诱导RAW264.7巨噬细胞AT1受体的表达,这种表达呈剂量依赖性和时间依赖性变化。 2.AT1受体介导了LPS和AngⅡ诱导RAW264.7巨噬细胞促炎性细胞因子TNF-α、IL-1β、IL-6和抗炎性细胞因子IL-10的产生,AngⅡ通过与AT1受体结合调节RAW264.7巨噬细胞ROS的产生。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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