细胞外信号调节激酶通路在香烟烟雾致肺血管重构中的调控机制

发布时间：2019-03-04 08:19

【摘要】：目的：探讨吸烟伴或不伴有慢性阻塞性肺疾病(chronic obstructive pulmonary disease, COPD)患者肺血管重塑中细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)的表达及意义。 方法：取48份(吸烟患COPD组16例、吸烟不患COPD组18例、不吸烟不患COPD组14例)手术切除的肺组织,HE染色观察肺血管重塑；用Western blot检测ERK及cyclinD1在肺动脉的表达；实时荧光RT-PCR检测cyclinD1mRNA的表达。 结果：与非吸烟对照组比较,吸烟不伴COPD组肺血管壁厚度和血管直径比值(%WT)明显增加,ERK及cyclinD1表达增加,差异有统计学意义(P0.01)。吸烟不伴COPD组与吸烟伴COPD组相比较,肺血管壁厚度和血管直径比值及ERK的表达差异无统计学意义。 结论：吸烟伴或不伴有COPD患者肺动脉ERK和cyclinD1的表达均较不吸烟者明显升高,ERK的高表达可能与香烟烟雾导致的肺血管重构有关。 目的：探讨细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)在香烟烟雾提取物(cigarette smoke extract, CSE)所致人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, HPASMCs)增殖中的作用。 方法：原代培养的HPASMCs予以CSE干预。细胞随机分为四组：正常对照组；5%CSE组；PD98059+5%CSE组；PD98059组。采用细胞计数及四甲基偶氮唑盐比色法(MTT)检测细胞的增殖能力。流式细胞术检测细胞周期的分布。用Western blot检测ERK及cyclinD1在HPASMCs的表达。实时荧光RT-PCR检测cyclinDl mRNA的表达。 结果：在5%CSE的刺激下,HPASMCs的ERK mRNA和蛋白水平均升高,并促进细胞增殖(P＜0.01)。PD98059抑制5%CSE诱导的细胞增殖(P＜0.01)。此外,PD98059明显抑制cyclinDl的mRNA和蛋白的表达,并导致细胞周期G1期停滞,进而减少HPASMCs的增殖(P0.01)。 结论：ERK在CSE诱导HPASMCs增殖中发挥重要作用,其部分原因可能是其上调cyclinD1的表达。 目的：探讨还原型辅酶Ⅱ(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶在香烟烟雾提取物(cigarette smoke extract, CSE)所致人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells, HPASMCs)增殖中的作用。 方法：原代培养的HPASMCs予以CSE干预。细胞随机分为四组：正常对照组；5%CSE组；apocynin+5%CSE组;apocynin组。采用细胞计数及四甲基偶氮唑盐比色法(MTT)检测细胞的增殖能力。流式细胞术检测细胞周期的分布。用Western blot检测NADPH氧化酶亚基p47phox及细胞外信号调节激酶(extracellular signal-regulated kinase, ERK)在HPASMCs的表达。实时荧光RT-PCR检测p47phox mRNA的表达。比色法检测丙二醛(malondiadehyde, MDA)表达。 结果：在5%CSE的刺激下,HPASMCs中的p47phox以及ERK表达水平升高,并促进细胞增殖(P＜0.01)。Apocynin抑制5%CSE诱导的细胞增殖(P＜0.01)。此外,apocynin明显抑制ERK的表达,并导致细胞周期G1期停滞,进而减少HPASMCs的增殖(P＜0.01)。 结论：NADPH氧化酶作为ERK的上游信号通路可能在CSE诱导HPASMCs增殖中发挥了重要的调控机制。
[Abstract]:Aim: to investigate the expression and significance of extracellular signal regulated kinase (extracellular signal-regulated kinase, ERK) in pulmonary vascular remodeling in patients with or without chronic obstructive pulmonary disease (chronic obstructive pulmonary disease, COPD). Methods: 48 surgically resected lung tissues (16 cases with COPD, 18 cases with COPD without smoking and 14 cases with no smoking without COPD) were taken to observe the pulmonary vascular remodeling by HE staining, and the expression of ERK and cyclinD1 in pulmonary artery were detected by Western blot. The expression of cyclinD1mRNA was detected by real-time fluorescence RT-PCR. Results: compared with the non-smoking control group, the pulmonary vascular wall thickness and vascular diameter ratio (% WT) and the expression of ERK and cyclinD1 were significantly increased in the non-smoking group without COPD (P0.01). There was no significant difference in pulmonary vascular wall thickness, vessel diameter ratio and ERK expression between smoking without COPD group and smoking with COPD group. Conclusion: the expression of ERK and cyclinD1 in pulmonary artery of smoking patients with or without COPD is significantly higher than that of non-smokers. The high expression of ERK may be related to pulmonary vascular remodeling induced by cigarette smoke. Aim: to investigate the role of extracellular signal regulated kinase (extracellular signal-regulated kinase, ERK) in the proliferation of human pulmonary artery smooth muscle cells (human pulmonary artery smooth muscle cells, HPASMCs) induced by cigarette smoke extract (cigarette smoke extract, CSE). Methods: primary cultured HPASMCs were treated with CSE. Cells were randomly divided into four groups: normal control group; 5%CSE group; PD98059 5%CSE group; PD98059 group. Cell count and tetramethylazo salt colorimetric assay (MTT) were used to detect the proliferation ability of the cells. Cell cycle distribution was detected by flow cytometry. The expression of ERK and cyclinD1 in HPASMCs was detected by Western blot. The expression of cyclinDl mRNA was detected by real-time fluorescence RT-PCR. Results: under the stimulation of 5%CSE, the levels of ERK mRNA and protein of HPASMCs increased and promoted the proliferation of cells (P < 0 01). PD98059 inhibited 5%CSE induced cell proliferation (P < 0 01). In addition, PD98059 significantly inhibited the expression of mRNA and protein in cyclinDl, and led to cell cycle arrest in G1 phase, thus reducing the proliferation of HPASMCs (P0.01). Conclusion: ERK plays an important role in the proliferation of HPASMCs induced by CSE, which may be due to its up-regulation of cyclinD1 expression. Aim: to investigate the role of reduced coenzyme 鈪，

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