α肾上腺素能受体激动对TLR4信号通路影响的研究
发布时间：2019-05-20 03:02
【摘要】：目的:研究α肾上腺素能受体激动对人外周血单核细胞TLR4信号通路及其中相关因子变化影响,探讨α肾上腺素能受体与TLR4的相互作用及其调控机制。 方法:采集健康青年人外周静脉血,进行离体实验。(一)观察不同剂量α受体激动剂去甲肾上腺素(Norepinephrine,NE)对人外周血单核细胞TLR4蛋白及mRNA、P38mRNA、NF-κBp65的影响及上清中炎症因子IL-6、IL-18的变化,选取10例健康青年人各抽取外周静脉血32ml,经EDTA抗凝后每份血均分为空白组、25μmol/L去甲肾上腺组、50μmol/L去甲肾上腺组、75μmol/L去甲肾上腺组,孵育18h后,用ELISA法测定血液离心后上清液中IL-6、IL-18的浓度,分离单核细胞后用流式细胞仪测定CD14+单核细胞TLR4阳性细胞百分率,提取RNA后采用RT-PCR测定TLR4mRNA及P38mRNA的表达,免疫组化法测定NF-κBp65蛋白的表达;(二)利用siRNA干扰技术使TLR4mRNA保持静默,观察用α受体激动剂NE刺激后相关因子浓度变化情况,选取10例健康人各抽取外周静脉血16ml,经EDTA抗凝后每份血均分为空白组、75μmol/L去甲肾上腺组、1nmol Scramble siRNA+0.24ml转染液组、1nmol TLR4 siRNA+0.24ml转染液组,转染18h后用ELISA法测定血液离心后上清液中IL-6、IL-18的浓度,分离单核细胞后爬片培养,免疫组化法测定NF-κBp65蛋白的表达。(三)利用不同阻滞剂分别阻断α受体、P38MAPK、PKA、PKC后,再用α受体激动剂NE刺激,观察TLR4蛋白表达量的变化,选取10例健康青年人各抽取外周静脉血6ml,经EDTA抗凝后每份血均分为25μmol/Lα受体阻滞剂酚妥拉明组、15μmol/L P38MAPK抑制剂SB 202190组、15μmol/L pKA抑制剂H89组、15μmol/L pKC抑制剂Go6983组,并设空白对照组、75μmol/L去甲肾上腺组,孵育0.5h后,再加入75μmol/LNE孵育18h后,采用流式细胞仪测定CD14+单核细胞TLR4阳性细胞百分率。 结果:1、应用不同浓度的α受体激动剂NE刺激人外周血单核细胞后,TLR4蛋白及mRNA、P38mRNA、核内NF-κBp65蛋白、上清中IL-6及IL-18的表达呈剂量依赖性升高:NE75组、NE50组分别与空白组及NE25组比较均明显升高,(p0.05,p0.01),NE75组与NE50组比较明显升高(p0.05),NE25组与空白组比较无明显升高(p0.05);2、TLR4mRNA干扰后α受体激动剂NE刺激血液离心后上清液中IL-6及IL-18、单核细胞NF-κB P65蛋白的表达为:Si+NE75组均明显低于空白组、Sc+ NE75组及NE75组(P0.05);3、分别阻断α受体、P38MAPK、PKA、PKC后,用有效浓度NE(75μmol/L)刺激外周血单核细胞,观察TLR4蛋白的表达量为:α-blocker组、P38组、PKA组与NE75组比较均明显降低(p0.05),各组与空白组比较未见明显差异(p0.05),PKC组与NE75组及空白组比较均明显增加(p0.05)。 结论: 1)α受体激动剂呈剂量依赖性诱导人外周血单核细胞TLR4信号通路激活,以75μmol/L去甲肾上腺素为最佳剂量; 2)α受体、P38MAPK、PKA、NF-κB P65介导了α受体激动剂诱导的TLR4信号通路激活。
[Abstract]:Aim: to study the effect of 伪 adrenergic receptor activation on TLR4 signaling pathway and its related factors in human peripheral blood monocytes, and to explore the interaction between 伪 adrenergic receptor and TLR4 and its regulatory mechanism. Methods: peripheral venous blood was collected from healthy young people and tested in vitro. (1) to observe the effects of different doses of 伪 receptor agonist norepinephrine (Norepinephrine,NE) on TLR4 protein and mRNA,P38mRNA,NF- 魏 Bp65 in human peripheral blood monocytes and the changes of inflammatory factor IL-6,IL-18 in the upper serum. Ten healthy young people were divided into blank group, 25 渭 mol / L norepinephrine group, 50 渭 mol / L norepinephrine group and 75 渭 mol / L norepinephrine group after 18 h incubation with 32 ml of peripheral venous blood collected from 10 healthy young people. After anticoagulant treatment with EDTA, each portion of blood was divided into blank group, 25 渭 mol / L noradrenalin group, 50 渭 mol / L noradrenalin group and 75 渭 mol / L noradrenalin group. The concentration of IL-6,IL-18 in the culture medium after blood centrifugation was determined by ELISA method, the percentage of TLR4 positive cells in CD14 monocytes was measured by flow cytometry after monocytes were isolated, and the expressions of TLR4mRNA and P38mRNA were detected by RT-PCR after RNA was extracted. The expression of NF- 魏 Bp65 protein was detected by immunohistochemistry. (2) using siRNA interference technique to keep TLR4mRNA silent, and observing the changes of related factors after stimulation with 伪 receptor agonist NE, 10 healthy people were selected to take 16 ml of peripheral venous blood. After EDTA anticoagulation, each blood was divided into blank group. 75 渭 mol / L norepinephrine group, 1nmol Scramble siRNA 0.24ml transfer solution group and 1nmol TLR4 siRNA 0.24ml transfection solution group. The concentration of IL-6,IL-18 in the culture medium after blood centrifugation was determined by ELISA method 18 hours later, and monocytes were isolated and cultured. The expression of NF- 魏 Bp65 protein was detected by immunohistochemistry. (3) using different blockers to block 伪 receptor, P38MapK, PKA, PKC, then stimulated by 伪 receptor agonist NE, the expression of TLR4 protein was observed. 6 ml of peripheral venous blood was taken from 10 healthy young people. After EDTA anticoagulant, each blood was divided into 25 渭 mol / L 伪 receptor blocker phentolamine group, 15 渭 mol / L P38MAPK inhibitor SB 202190 group, 15 渭 mol / L pKA inhibitor H89 group, 15 渭 mol / L pKC inhibitor Go6983 group, and blank control group, 75 渭 mol / L norepinephrine group. After 0.5 h incubation, then incubated with 75 渭 mol / LNE for 18 h, the percentage of TLR4 positive cells in CD14 monocytes was measured by flow cytometry. Results: 1. After stimulation of human peripheral blood monocytes with different concentrations of 伪 receptor agonist NE, the expression of TLR4 protein and NF- 魏 Bp65 protein in mRNA,P38mRNA, nucleus increased in a dose-dependent manner: in NE75 group, NE50 group was significantly higher than blank group and NE25 group (p0.05, p0.01), NE75 group was significantly higher than NE50 group (p0.05), NE25 group was not significantly higher than blank group (p0.05). 2. The expression of IL-6 and IL-18, monocyte NF- 魏 B p65 protein in IL-6 and IL-18, monocytes stimulated by 伪 receptor agonist NE after TLR4 mRNA interference was significantly lower in Si NE75 group than in blank group, Sc NE75 group and NE75 group (P 0.05). 3. After blocking 伪 receptor, P38MapK, PKA, PKC, peripheral blood monocytes were stimulated with effective concentration of NE (75 渭 mol / L). The expression of TLR4 protein in 伪-blocker group, P38 group and PKA group was significantly lower than that in NE75 group (p0.05). There was no significant difference between each group and the blank group (p0.05), PKC group was significantly higher than that of the NE75 group and the blank group (p0.05). Conclusion: 1) 伪 receptor agonists induce the activation of TLR4 signaling pathway in human peripheral blood monocytes in a dose-dependent manner, and 75 渭 mol / L norepinephrine is the best dose. 2) 伪 receptor, P38mapK, PKA, NF-魏 B p65 mediated the activation of TLR4 signal pathway induced by 伪 receptor agonist.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
本文编号：2481291
[Abstract]:Aim: to study the effect of 伪 adrenergic receptor activation on TLR4 signaling pathway and its related factors in human peripheral blood monocytes, and to explore the interaction between 伪 adrenergic receptor and TLR4 and its regulatory mechanism. Methods: peripheral venous blood was collected from healthy young people and tested in vitro. (1) to observe the effects of different doses of 伪 receptor agonist norepinephrine (Norepinephrine,NE) on TLR4 protein and mRNA,P38mRNA,NF- 魏 Bp65 in human peripheral blood monocytes and the changes of inflammatory factor IL-6,IL-18 in the upper serum. Ten healthy young people were divided into blank group, 25 渭 mol / L norepinephrine group, 50 渭 mol / L norepinephrine group and 75 渭 mol / L norepinephrine group after 18 h incubation with 32 ml of peripheral venous blood collected from 10 healthy young people. After anticoagulant treatment with EDTA, each portion of blood was divided into blank group, 25 渭 mol / L noradrenalin group, 50 渭 mol / L noradrenalin group and 75 渭 mol / L noradrenalin group. The concentration of IL-6,IL-18 in the culture medium after blood centrifugation was determined by ELISA method, the percentage of TLR4 positive cells in CD14 monocytes was measured by flow cytometry after monocytes were isolated, and the expressions of TLR4mRNA and P38mRNA were detected by RT-PCR after RNA was extracted. The expression of NF- 魏 Bp65 protein was detected by immunohistochemistry. (2) using siRNA interference technique to keep TLR4mRNA silent, and observing the changes of related factors after stimulation with 伪 receptor agonist NE, 10 healthy people were selected to take 16 ml of peripheral venous blood. After EDTA anticoagulation, each blood was divided into blank group. 75 渭 mol / L norepinephrine group, 1nmol Scramble siRNA 0.24ml transfer solution group and 1nmol TLR4 siRNA 0.24ml transfection solution group. The concentration of IL-6,IL-18 in the culture medium after blood centrifugation was determined by ELISA method 18 hours later, and monocytes were isolated and cultured. The expression of NF- 魏 Bp65 protein was detected by immunohistochemistry. (3) using different blockers to block 伪 receptor, P38MapK, PKA, PKC, then stimulated by 伪 receptor agonist NE, the expression of TLR4 protein was observed. 6 ml of peripheral venous blood was taken from 10 healthy young people. After EDTA anticoagulant, each blood was divided into 25 渭 mol / L 伪 receptor blocker phentolamine group, 15 渭 mol / L P38MAPK inhibitor SB 202190 group, 15 渭 mol / L pKA inhibitor H89 group, 15 渭 mol / L pKC inhibitor Go6983 group, and blank control group, 75 渭 mol / L norepinephrine group. After 0.5 h incubation, then incubated with 75 渭 mol / LNE for 18 h, the percentage of TLR4 positive cells in CD14 monocytes was measured by flow cytometry. Results: 1. After stimulation of human peripheral blood monocytes with different concentrations of 伪 receptor agonist NE, the expression of TLR4 protein and NF- 魏 Bp65 protein in mRNA,P38mRNA, nucleus increased in a dose-dependent manner: in NE75 group, NE50 group was significantly higher than blank group and NE25 group (p0.05, p0.01), NE75 group was significantly higher than NE50 group (p0.05), NE25 group was not significantly higher than blank group (p0.05). 2. The expression of IL-6 and IL-18, monocyte NF- 魏 B p65 protein in IL-6 and IL-18, monocytes stimulated by 伪 receptor agonist NE after TLR4 mRNA interference was significantly lower in Si NE75 group than in blank group, Sc NE75 group and NE75 group (P 0.05). 3. After blocking 伪 receptor, P38MapK, PKA, PKC, peripheral blood monocytes were stimulated with effective concentration of NE (75 渭 mol / L). The expression of TLR4 protein in 伪-blocker group, P38 group and PKA group was significantly lower than that in NE75 group (p0.05). There was no significant difference between each group and the blank group (p0.05), PKC group was significantly higher than that of the NE75 group and the blank group (p0.05). Conclusion: 1) 伪 receptor agonists induce the activation of TLR4 signaling pathway in human peripheral blood monocytes in a dose-dependent manner, and 75 渭 mol / L norepinephrine is the best dose. 2) 伪 receptor, P38mapK, PKA, NF-魏 B p65 mediated the activation of TLR4 signal pathway induced by 伪 receptor agonist.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
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