ICAM-1和CX3CL1对内皮细胞骨架的影响及其机制的探讨
发布时间：2019-06-05 22:25
【摘要】：目的：观察ICAM-1和CX3CL1对人脐静脉内皮细胞(HUVECs)骨架的影响,探讨其可能的机制。方法：(1)观察ICAM-1和CX3CL1对HUVECs的骨架蛋白F-actin的影响：HUVECs饥饿处理12h；①10 μM Fg刺激HUVECs30 min后,1 nM ICAM-1分别刺激HUVECs0、30、60、120、180 min； ②10 nMCX3CL1分别刺激HUVECs0、30、60、120、180 min 4%多聚甲醛固定细胞后,用Alexafluor(?) 488 phalloidin对细胞骨架蛋白F-actin进行荧光染色,荧光显微镜下观察细胞F-actin形态和分布情况。(2)检测ICAM-1和CX3CL1对HUVECs的MAPK信号通路的影响：KUVECs饥饿处理12h；ο 10 μM Fg刺激HUVECs30 min后1 nMICAM-1分别刺激HUVECs 0,5、、15、30、60 min； ②10 nM CX3CL1分别刺激HUVECs 0、1、5、15、30 min;③ 5 μg/mL抗ICAM-1+10 μM Fg刺激细胞30 min后,1 nM ICAM-1刺激细胞5 min、15min和15 min、30 min；④ 5μg/mL抗CX3CR1抗体刺激内皮细胞30 min后,10nM CX3CL1刺激细胞1min和5 min；提取细胞总蛋白,Western blot检测磷酸化p38、ERK1/2和JNK1/2和总p38、ERK1/2和JNK1/2的表达水平。(3)探讨ICAM-1和CX3CL1介导的HUVECs骨架改变的机制：HUVECs饥饿处理12 h；①30 gM的(SB203580p38的特异性抑制剂)、PD98059(ERK1/2的特异性抑制剂)和SP600125(JNK1/2的特异性拮抗剂)分别刺激内皮细胞30 min后,10 μM Fg刺激HUVECs 30 min,随后1 nMICAM-1在刺激HUVECs180 min;②30 μM SB203580、PD98059和SP600125分别刺激HUVECs1h后,10 nM CX3CL1 刺激 HUVECs120 min；固定细胞后,对细胞进行荧光染色,荧光显微镜下观察细胞F-actin形态和分布情况。结果：(1)空白对照的HUVECs的细胞周边的F-actin形成光滑而连续的外周致密带。1 nM ICAM-1刺激HUVECs30 min后,细胞没有明显变化；60 min后胞质内出现少量应力纤维,细胞膜外周的F-actin成锯齿状,致密带被破坏；120 min后,细胞周边的F-actin基本消失,细胞质内出现大量纤细的应力纤维；180 mim后细胞质内出现密集的贯穿整个细胞的应力纤维,细胞变形。10 nM CX3CL1刺激HUVECs 30min后胞质内出现少量应力纤维,细胞外周的F-actin成锯齿状,致密带被破坏；60 min后,细胞质内出现大量纤细的应力纤维,而细胞周边的F-actin基本消失；120 min后细胞质内出现密集的贯穿整个细胞的应力纤维,细胞变形；180 min后在胞质内的应力纤维变短而稀疏。(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对照的细胞,15 min后磷酸化ERK1/2和JNK1/2的表达水平达峰值,而磷酸化p38的的表达水平在30 min后达峰值；10 nM CX3CL1刺激HUVECs1 min后p38、ERK1/2和JNK1/2磷酸化水平高于空白对照的细胞,三者的磷酸化水平均在5 min后达峰值；5μg/mL抗1nM ICAM-1抗体抑制]CAM-1介导的p38、ERK1/2和JNK1/2磷酸化水平的上调；5 μg/mL抗CX3CR1抗体抑制10 nMCX3CL1介导的的p38、ERK1/2和JNK1/2磷酸化水平的上调。(3)30μMSB203580提前干预后,lnM ICAM-1刺激内皮细胞后引起的F-actin的重排及应力纤维的形成被抑制,30 μM SB203580和PD98059提前干预后,10 nM CX3CL1刺激内皮细胞后引起的F-actin的重排及应力纤维的形成被抑制。结论：(1)ICAM-1可能以时间依赖方式通过激活p38信号通路介导HUVECs细胞骨架的重构；(2) CX3CL1可能以时间依赖的方式通过p38和ERK1/2信号通路介导HUVECs细胞骨架的重构。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R593.2
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