α腎上腺素能受體激動對TLR4信號通路影響的研究
發(fā)布時間：2019-05-20 03:02
【摘要】：目的:研究α腎上腺素能受體激動對人外周血單核細(xì)胞TLR4信號通路及其中相關(guān)因子變化影響,探討α腎上腺素能受體與TLR4的相互作用及其調(diào)控機制。 方法:采集健康青年人外周靜脈血,進(jìn)行離體實驗。(一)觀察不同劑量α受體激動劑去甲腎上腺素(Norepinephrine,NE)對人外周血單核細(xì)胞TLR4蛋白及mRNA、P38mRNA、NF-κBp65的影響及上清中炎癥因子IL-6、IL-18的變化,選取10例健康青年人各抽取外周靜脈血32ml,經(jīng)EDTA抗凝后每份血均分為空白組、25μmol/L去甲腎上腺組、50μmol/L去甲腎上腺組、75μmol/L去甲腎上腺組,孵育18h后,用ELISA法測定血液離心后上清液中IL-6、IL-18的濃度,分離單核細(xì)胞后用流式細(xì)胞儀測定CD14+單核細(xì)胞TLR4陽性細(xì)胞百分率,提取RNA后采用RT-PCR測定TLR4mRNA及P38mRNA的表達(dá),免疫組化法測定NF-κBp65蛋白的表達(dá);(二)利用siRNA干擾技術(shù)使TLR4mRNA保持靜默,觀察用α受體激動劑NE刺激后相關(guān)因子濃度變化情況,選取10例健康人各抽取外周靜脈血16ml,經(jīng)EDTA抗凝后每份血均分為空白組、75μmol/L去甲腎上腺組、1nmol Scramble siRNA+0.24ml轉(zhuǎn)染液組、1nmol TLR4 siRNA+0.24ml轉(zhuǎn)染液組,轉(zhuǎn)染18h后用ELISA法測定血液離心后上清液中IL-6、IL-18的濃度,分離單核細(xì)胞后爬片培養(yǎng),免疫組化法測定NF-κBp65蛋白的表達(dá)。(三)利用不同阻滯劑分別阻斷α受體、P38MAPK、PKA、PKC后,再用α受體激動劑NE刺激,觀察TLR4蛋白表達(dá)量的變化,選取10例健康青年人各抽取外周靜脈血6ml,經(jīng)EDTA抗凝后每份血均分為25μmol/Lα受體阻滯劑酚妥拉明組、15μmol/L P38MAPK抑制劑SB 202190組、15μmol/L pKA抑制劑H89組、15μmol/L pKC抑制劑Go6983組,并設(shè)空白對照組、75μmol/L去甲腎上腺組,孵育0.5h后,再加入75μmol/LNE孵育18h后,采用流式細(xì)胞儀測定CD14+單核細(xì)胞TLR4陽性細(xì)胞百分率。 結(jié)果:1、應(yīng)用不同濃度的α受體激動劑NE刺激人外周血單核細(xì)胞后,TLR4蛋白及mRNA、P38mRNA、核內(nèi)NF-κBp65蛋白、上清中IL-6及IL-18的表達(dá)呈劑量依賴性升高:NE75組、NE50組分別與空白組及NE25組比較均明顯升高,(p0.05,p0.01),NE75組與NE50組比較明顯升高(p0.05),NE25組與空白組比較無明顯升高(p0.05);2、TLR4mRNA干擾后α受體激動劑NE刺激血液離心后上清液中IL-6及IL-18、單核細(xì)胞NF-κB P65蛋白的表達(dá)為:Si+NE75組均明顯低于空白組、Sc+ NE75組及NE75組(P0.05);3、分別阻斷α受體、P38MAPK、PKA、PKC后,用有效濃度NE(75μmol/L)刺激外周血單核細(xì)胞,觀察TLR4蛋白的表達(dá)量為:α-blocker組、P38組、PKA組與NE75組比較均明顯降低(p0.05),各組與空白組比較未見明顯差異(p0.05),PKC組與NE75組及空白組比較均明顯增加(p0.05)。 結(jié)論: 1)α受體激動劑呈劑量依賴性誘導(dǎo)人外周血單核細(xì)胞TLR4信號通路激活,以75μmol/L去甲腎上腺素為最佳劑量; 2)α受體、P38MAPK、PKA、NF-κB P65介導(dǎo)了α受體激動劑誘導(dǎo)的TLR4信號通路激活。
[Abstract]:Aim: to study the effect of 偽 adrenergic receptor activation on TLR4 signaling pathway and its related factors in human peripheral blood monocytes, and to explore the interaction between 偽 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 偽 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 偽 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 偽 receptor, P38MapK, PKA, PKC, then stimulated by 偽 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 偽 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 偽 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 偽 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 偽 receptor, P38MapK, PKA, PKC, peripheral blood monocytes were stimulated with effective concentration of NE (75 渭 mol / L). The expression of TLR4 protein in 偽-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) 偽 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) 偽 receptor, P38mapK, PKA, NF-魏 B p65 mediated the activation of TLR4 signal pathway induced by 偽 receptor agonist.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
本文編號：2481291
[Abstract]:Aim: to study the effect of 偽 adrenergic receptor activation on TLR4 signaling pathway and its related factors in human peripheral blood monocytes, and to explore the interaction between 偽 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 偽 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 偽 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 偽 receptor, P38MapK, PKA, PKC, then stimulated by 偽 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 偽 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 偽 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 偽 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 偽 receptor, P38MapK, PKA, PKC, peripheral blood monocytes were stimulated with effective concentration of NE (75 渭 mol / L). The expression of TLR4 protein in 偽-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) 偽 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) 偽 receptor, P38mapK, PKA, NF-魏 B p65 mediated the activation of TLR4 signal pathway induced by 偽 receptor agonist.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
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