ICAM-1和CX3CL1對(duì)內(nèi)皮細(xì)胞骨架的影響及其機(jī)制的探討
發(fā)布時(shí)間：2019-06-05 22:25
【摘要】：目的：觀察ICAM-1和CX3CL1對(duì)人臍靜脈內(nèi)皮細(xì)胞(HUVECs)骨架的影響,探討其可能的機(jī)制。方法：(1)觀察ICAM-1和CX3CL1對(duì)HUVECs的骨架蛋白F-actin的影響：HUVECs饑餓處理12h；①10 μM Fg刺激HUVECs30 min后,1 nM ICAM-1分別刺激HUVECs0、30、60、120、180 min； ②10 nMCX3CL1分別刺激HUVECs0、30、60、120、180 min 4%多聚甲醛固定細(xì)胞后,用Alexafluor(?) 488 phalloidin對(duì)細(xì)胞骨架蛋白F-actin進(jìn)行熒光染色,熒光顯微鏡下觀察細(xì)胞F-actin形態(tài)和分布情況。(2)檢測(cè)ICAM-1和CX3CL1對(duì)HUVECs的MAPK信號(hào)通路的影響：KUVECs饑餓處理12h；ο 10 μM Fg刺激HUVECs30 min后1 nMICAM-1分別刺激HUVECs 0,5、、15、30、60 min； ②10 nM CX3CL1分別刺激HUVECs 0、1、5、15、30 min;③ 5 μg/mL抗ICAM-1+10 μM Fg刺激細(xì)胞30 min后,1 nM ICAM-1刺激細(xì)胞5 min、15min和15 min、30 min；④ 5μg/mL抗CX3CR1抗體刺激內(nèi)皮細(xì)胞30 min后,10nM CX3CL1刺激細(xì)胞1min和5 min；提取細(xì)胞總蛋白,Western blot檢測(cè)磷酸化p38、ERK1/2和JNK1/2和總p38、ERK1/2和JNK1/2的表達(dá)水平。(3)探討ICAM-1和CX3CL1介導(dǎo)的HUVECs骨架改變的機(jī)制：HUVECs饑餓處理12 h；①30 gM的(SB203580p38的特異性抑制劑)、PD98059(ERK1/2的特異性抑制劑)和SP600125(JNK1/2的特異性拮抗劑)分別刺激內(nèi)皮細(xì)胞30 min后,10 μM Fg刺激HUVECs 30 min,隨后1 nMICAM-1在刺激HUVECs180 min;②30 μM SB203580、PD98059和SP600125分別刺激HUVECs1h后,10 nM CX3CL1 刺激 HUVECs120 min；固定細(xì)胞后,對(duì)細(xì)胞進(jìn)行熒光染色,熒光顯微鏡下觀察細(xì)胞F-actin形態(tài)和分布情況。結(jié)果：(1)空白對(duì)照的HUVECs的細(xì)胞周邊的F-actin形成光滑而連續(xù)的外周致密帶。1 nM ICAM-1刺激HUVECs30 min后,細(xì)胞沒(méi)有明顯變化；60 min后胞質(zhì)內(nèi)出現(xiàn)少量應(yīng)力纖維,細(xì)胞膜外周的F-actin成鋸齒狀,致密帶被破壞；120 min后,細(xì)胞周邊的F-actin基本消失,細(xì)胞質(zhì)內(nèi)出現(xiàn)大量纖細(xì)的應(yīng)力纖維；180 mim后細(xì)胞質(zhì)內(nèi)出現(xiàn)密集的貫穿整個(gè)細(xì)胞的應(yīng)力纖維,細(xì)胞變形。10 nM CX3CL1刺激HUVECs 30min后胞質(zhì)內(nèi)出現(xiàn)少量應(yīng)力纖維,細(xì)胞外周的F-actin成鋸齒狀,致密帶被破壞；60 min后,細(xì)胞質(zhì)內(nèi)出現(xiàn)大量纖細(xì)的應(yīng)力纖維,而細(xì)胞周邊的F-actin基本消失；120 min后細(xì)胞質(zhì)內(nèi)出現(xiàn)密集的貫穿整個(gè)細(xì)胞的應(yīng)力纖維,細(xì)胞變形；180 min后在胞質(zhì)內(nèi)的應(yīng)力纖維變短而稀疏。(2) 1 nM ICAM-1刺激HUVECs5 min后p38、ERK1/2和JNK1/2磷酸化水平高于空白對(duì)照的細(xì)胞,15 min后磷酸化ERK1/2和JNK1/2的表達(dá)水平達(dá)峰值,而磷酸化p38的的表達(dá)水平在30 min后達(dá)峰值；10 nM CX3CL1刺激HUVECs1 min后p38、ERK1/2和JNK1/2磷酸化水平高于空白對(duì)照的細(xì)胞,三者的磷酸化水平均在5 min后達(dá)峰值；5μg/mL抗1nM ICAM-1抗體抑制]CAM-1介導(dǎo)的p38、ERK1/2和JNK1/2磷酸化水平的上調(diào)；5 μg/mL抗CX3CR1抗體抑制10 nMCX3CL1介導(dǎo)的的p38、ERK1/2和JNK1/2磷酸化水平的上調(diào)。(3)30μMSB203580提前干預(yù)后,lnM ICAM-1刺激內(nèi)皮細(xì)胞后引起的F-actin的重排及應(yīng)力纖維的形成被抑制,30 μM SB203580和PD98059提前干預(yù)后,10 nM CX3CL1刺激內(nèi)皮細(xì)胞后引起的F-actin的重排及應(yīng)力纖維的形成被抑制。結(jié)論：(1)ICAM-1可能以時(shí)間依賴(lài)方式通過(guò)激活p38信號(hào)通路介導(dǎo)HUVECs細(xì)胞骨架的重構(gòu)；(2) CX3CL1可能以時(shí)間依賴(lài)的方式通過(guò)p38和ERK1/2信號(hào)通路介導(dǎo)HUVECs細(xì)胞骨架的重構(gòu)。
[Abstract]:Objective: To observe the effect of ICAM-1 and CX3CL1 on the skeleton of human umbilical vein endothelial cells (HUVECs). Methods: (1) The effects of ICAM-1 and CX3CL1 on the F-actin of HUVECs were observed: HUVECs starvation treatment for 12 h; after 10. m u.M Fg of HUVECs for 30 min,1 nM of ICAM-1 stimulated HUVECs 0,30,60,120,180 min, and 10 nMCX3CL1 respectively stimulated HUVECs 0,30,60,120,180 min 4% paraformaldehyde fixed cells, and then used Alexafluor (? The morphology and distribution of F-actin were observed under the fluorescence microscope. (2) The effects of ICAM-1 and CX3CL1 on the MAPK signal pathway of HUVECs were detected: KUVECs starvation treatment for 12 h, and stimulation of HUVECs 0,5,15,30,60 min after HUVECs 30 min, and 10 nM CX3CL1 respectively stimulated HUVECs 0,1,5,15,30 min. After 30 min,1 nM ICAM-1 stimulated the cells for 5 min,15 min and 15 min. The expression levels of phosphorylated p38, ERK1/2 and JNK1/2 and total p38, ERK1/2 and JNK1/2 were detected by Western blot. (3) The mechanism of the change of HUVECs skeleton mediated by ICAM-1 and CX3CL1 was discussed: HUVECs starvation treatment for 12 h,30 gM (specific inhibitor of SB203580p38), PD98059 (specific inhibitor of ERK1/2) and SP600125 (specific antagonist of JNK1/2), respectively. Subsequently,1 nMCAM-1 stimulated HUVECs180 min;30. m u.M SB203580, PD98059 and SP600125 respectively stimulated HUVECs1h, and 10 nM CX3CL1 stimulated HUVECs120 min; after the cells were fixed, the cells were stained with fluorescence, and the morphology and distribution of F-actin were observed under a fluorescence microscope. Results: (1) The F-actin at the periphery of the cells of the HUVECs of the blank control group was smooth and continuous with a continuous peripheral compact band. After the stimulation of the HUVECs for 30 min, the cells did not change significantly; after 60 min, a small amount of stress fibers appeared in the cytoplasm, and the F-actin at the periphery of the cell membrane was serrated, and the dense band was destroyed; after 120 min, The F-actin in the periphery of the cell disappeared substantially, and a large number of fine stress fibers appeared in the cytoplasm, and the stress fibers and the cells of the whole cell appeared in the cytoplasm after 180 milm. The 10 nM CX3CL1 stimulated the presence of a small amount of stress fibers in the cytoplasm of the HUVECs for 30 min, and the F-actin in the outer periphery of the cells was serrated. The dense band was destroyed; after 60 min, a large number of fine stress fibers appeared in the cytoplasm, and the F-actin in the periphery of the cells disappeared substantially; after 120 min, a dense stress fiber penetrating the whole cell was observed, and the cells were deformed; and the stress fiber in the cytoplasm after 180 min was short and sparse. (2) The expression level of p38, ERK1/2 and JNK1/2 in p38, ERK1/2 and JNK1/2 was higher than that of blank control after 1 nM ICAM-1 stimulation, and the expression level of phosphorylated ERK1/2 and JNK1/2 reached the peak after 15 min, while the expression level of phosphorylated p38 reached the peak after 30 min. p38 after 10 nM CX3CL1 stimulated HUVECs1 min. The phosphorylation levels of ERK1/2 and JNK1/2 were higher than that of the blank control, and the phosphorylation levels of the three were up to peak at 5 min;5. mu.g/ mL of anti-1 nM ICAM-1 antibody inhibition] CAM-1-mediated upregulation of p38, ERK1/2 and JNK1/2 phosphorylation levels;5. mu.g/ mL anti-CX3CR1 antibody inhibited p38 mediated by 10 nMCX3CL1, Up-regulation of ERK1/2 and JNK1/2 phosphorylation levels. (3) After the early intervention of 30 & mu; MSB203580, the rearrangement of F-actin and the formation of stress fibers induced by lnM ICAM-1 stimulated the endothelial cells were inhibited, and the rearrangement of F-actin and the formation of stress fibers of 10 nM CX3CL1 were inhibited after the early intervention of 30. m u.M SB203580 and PD98059. Conclusion: (1) ICAM-1 may mediate the remodeling of HUVECs cytoskeleton by activating p38 signaling pathway in time-dependent manner; (2) CX3CL1 may mediate the reconstruction of HUVECs cytoskeleton in time-dependent manner through p38 and ERK1/2 signaling pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R593.2
[Abstract]:Objective: To observe the effect of ICAM-1 and CX3CL1 on the skeleton of human umbilical vein endothelial cells (HUVECs). Methods: (1) The effects of ICAM-1 and CX3CL1 on the F-actin of HUVECs were observed: HUVECs starvation treatment for 12 h; after 10. m u.M Fg of HUVECs for 30 min,1 nM of ICAM-1 stimulated HUVECs 0,30,60,120,180 min, and 10 nMCX3CL1 respectively stimulated HUVECs 0,30,60,120,180 min 4% paraformaldehyde fixed cells, and then used Alexafluor (? The morphology and distribution of F-actin were observed under the fluorescence microscope. (2) The effects of ICAM-1 and CX3CL1 on the MAPK signal pathway of HUVECs were detected: KUVECs starvation treatment for 12 h, and stimulation of HUVECs 0,5,15,30,60 min after HUVECs 30 min, and 10 nM CX3CL1 respectively stimulated HUVECs 0,1,5,15,30 min. After 30 min,1 nM ICAM-1 stimulated the cells for 5 min,15 min and 15 min. The expression levels of phosphorylated p38, ERK1/2 and JNK1/2 and total p38, ERK1/2 and JNK1/2 were detected by Western blot. (3) The mechanism of the change of HUVECs skeleton mediated by ICAM-1 and CX3CL1 was discussed: HUVECs starvation treatment for 12 h,30 gM (specific inhibitor of SB203580p38), PD98059 (specific inhibitor of ERK1/2) and SP600125 (specific antagonist of JNK1/2), respectively. Subsequently,1 nMCAM-1 stimulated HUVECs180 min;30. m u.M SB203580, PD98059 and SP600125 respectively stimulated HUVECs1h, and 10 nM CX3CL1 stimulated HUVECs120 min; after the cells were fixed, the cells were stained with fluorescence, and the morphology and distribution of F-actin were observed under a fluorescence microscope. Results: (1) The F-actin at the periphery of the cells of the HUVECs of the blank control group was smooth and continuous with a continuous peripheral compact band. After the stimulation of the HUVECs for 30 min, the cells did not change significantly; after 60 min, a small amount of stress fibers appeared in the cytoplasm, and the F-actin at the periphery of the cell membrane was serrated, and the dense band was destroyed; after 120 min, The F-actin in the periphery of the cell disappeared substantially, and a large number of fine stress fibers appeared in the cytoplasm, and the stress fibers and the cells of the whole cell appeared in the cytoplasm after 180 milm. The 10 nM CX3CL1 stimulated the presence of a small amount of stress fibers in the cytoplasm of the HUVECs for 30 min, and the F-actin in the outer periphery of the cells was serrated. The dense band was destroyed; after 60 min, a large number of fine stress fibers appeared in the cytoplasm, and the F-actin in the periphery of the cells disappeared substantially; after 120 min, a dense stress fiber penetrating the whole cell was observed, and the cells were deformed; and the stress fiber in the cytoplasm after 180 min was short and sparse. (2) The expression level of p38, ERK1/2 and JNK1/2 in p38, ERK1/2 and JNK1/2 was higher than that of blank control after 1 nM ICAM-1 stimulation, and the expression level of phosphorylated ERK1/2 and JNK1/2 reached the peak after 15 min, while the expression level of phosphorylated p38 reached the peak after 30 min. p38 after 10 nM CX3CL1 stimulated HUVECs1 min. The phosphorylation levels of ERK1/2 and JNK1/2 were higher than that of the blank control, and the phosphorylation levels of the three were up to peak at 5 min;5. mu.g/ mL of anti-1 nM ICAM-1 antibody inhibition] CAM-1-mediated upregulation of p38, ERK1/2 and JNK1/2 phosphorylation levels;5. mu.g/ mL anti-CX3CR1 antibody inhibited p38 mediated by 10 nMCX3CL1, Up-regulation of ERK1/2 and JNK1/2 phosphorylation levels. (3) After the early intervention of 30 & mu; MSB203580, the rearrangement of F-actin and the formation of stress fibers induced by lnM ICAM-1 stimulated the endothelial cells were inhibited, and the rearrangement of F-actin and the formation of stress fibers of 10 nM CX3CL1 were inhibited after the early intervention of 30. m u.M SB203580 and PD98059. Conclusion: (1) ICAM-1 may mediate the remodeling of HUVECs cytoskeleton by activating p38 signaling pathway in time-dependent manner; (2) CX3CL1 may mediate the reconstruction of HUVECs cytoskeleton in time-dependent manner through p38 and ERK1/2 signaling pathway.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R593.2
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