橙皮素对血小板源性生长因子-BB所致肺动脉平滑肌细胞增殖的作用研究
发布时间：2019-03-16 12:47
【摘要】：背景：肺动脉高压(Pulmonary arterial hypertension, PAH)是指处于安静状态下肺动脉平均压25mmHg。它是由不同病因和发病机制引起的在多种临床疾病中均可出现的以肺血管阻力持续升高为特点的临床综合征。研究认为,肺动脉高压时肺血管可以出现下述诸如内膜增生,中膜肥厚,外膜增殖、纤维变性,小血管闭塞,血栓形成,炎症细胞浸润等等改变。在这之中,肺动脉平滑肌细胞(pulmonary aterial smooth muscle cells, PASMCs)的异常过度的增殖在肺血管的重构中起到关键作用。长期持续的肺动脉高压最终会导致右心室衰竭甚至死亡,尽管本病对健康水平可造成严重影响,但目前肺动脉高压的常用药物均只能改善患者的症状而不能延缓其发展进程。因此寻找能够阻滞甚至逆转肺动脉平滑肌细胞异常增殖的物质,探索新的治疗手段,对提高肺动脉高压患者的治疗效果和生活质量具有重要的价值。目的：建立血小板源性生长因子(platelet-derived growth factor, PDGF)-BB诱导的原代大鼠肺动脉平滑肌细胞的增殖细胞模型,并通过使用不同浓度的橙皮素进行干预,探讨橙皮素对肺动脉平滑肌细胞增殖的影响及其相关机制,为肺血管重构的防治寻找新药物。方法：选择体重处于150-200 g的SD大鼠,分离肺动脉,选用0.2%I型胶原酶消化法分离肺动脉平滑肌细胞。应用20ng/ml PDGF-BB诱导肺动脉平滑肌细胞增殖创建细胞模型。应用不同浓度橙皮素干预(12.5、25、50、100μmol/L),通过CCK-8及BRDU法于12、24、48小时检测PASMCs的增殖和DNA合成；不同浓度橙皮素(12.5、25、50、100μmol/L)处理PASMCs于12、24、48小时后通过0.4%浓度的台盼蓝染色法检测细胞存活率；予100μmol/L橙皮素干预PDGF-BB刺激肺动脉平滑肌细胞24小时后,采用流式细胞仪分析细胞周期,实时定量PCR (RT-PCR)检测细胞周期相关性蛋白D1,细胞周期相关性蛋白E,CDK2.CDK4和细胞周期激酶抑制蛋白p27等细胞周期相关调控蛋白mRNA的表达；用100μmol/L橙皮素干预PDGF-BB刺激的肺动脉平滑肌细胞,于作用后5、10、15分钟应用Western Blotting的方法来检测比较总的以及磷酸化的ERK1/2,p38, JNK, AKT,糖原合酶激酶3β(GSK3β)。结果：CCK-8和BRDU法检测结果表明橙皮素能够抑制PDGF-BB刺激的PASMCs增殖及DNA的合成,并且这种抑制作用具有时间依赖性和浓度依赖性。台盼蓝染色的结果表明本实验应用的各浓度组(12.5,25,50, 100μmol/L)橙皮素对处理过后的PASMCs其存活率没有显著的毒性作用；流式细胞仪分析结果表明100μmol/L橙皮素能够使G0/G1期细胞增多(62.0±0.6%-69.7±0.3%),S期细胞减少(25.7±0.9%-17.7±1.2%),抑制细胞周期于G0/G1-S期,Real Time PCR结果表明橙皮素对细胞周期的抑制作用与下调细胞周期蛋白D1,细胞周期蛋白E,CDK2, CDK4和上调P27的mRNA表达有关；进一步研究表明100μmol/L橙皮素能够抑制AKT/GSK3β和p38信号通路的磷酸化,而对ERK1/2和JNK信号通路无明显影响。结论：橙皮素可以抑制PDGF-BB诱导的PASMCs增殖,橙皮素的抑制作用是通过下调细胞周期相关性蛋白D1,细胞周期相关性蛋白E,CDK2,CDK4及上调细胞周期激酶抑制性蛋白P27的mRNA表达,将细胞周期阻滞于G0/G1-S期来实现,进一步研究提示这种抑制作用与抑制AKT/GSK3β和p38信号通路的磷酸化水平有关。橙皮素有潜力成为临床治疗肺动脉高压的药物。
[Abstract]:Background: Pulmonary hypertension (PAH) is the mean pressure of the pulmonary artery in a quiet state of 25 mmHg. It is a clinical syndrome characterized by the continuous increase of pulmonary vascular resistance that can occur in various clinical diseases caused by different etiologies and pathogenesis. The study found that the pulmonary vessels in the pulmonary artery at high pressure could be changed as described below, such as intimal hyperplasia, media hypertrophy, adventitia proliferation, fibrosis, small vessel occlusion, thrombosis, inflammatory cell infiltration, and the like. In this, the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a key role in the reconstruction of pulmonary vessels. Long-term persistent pulmonary hypertension may eventually lead to right ventricular failure or even death, although this disease may have a serious effect on the level of health, but the common use of high pressure in the pulmonary artery can only improve the symptoms of the patient and not delay the development process. Therefore, it is of great value to find a substance capable of blocking and even reversing the abnormal proliferation of pulmonary artery smooth muscle cells, and to explore new therapeutic means and to improve the therapeutic effect and quality of life of patients with pulmonary hypertension. Objective: To establish a cell model of the proliferation of primary rat pulmonary artery smooth muscle cells induced by platelet-derived growth factor (PDGF)-BB and to study the effect of orange peel on the proliferation of pulmonary artery smooth muscle cells and its related mechanisms by using different concentrations of orange peel. To search for new drugs for the prevention and treatment of pulmonary vascular remodeling. Methods: SD rats weighing 150-200 g were selected and the pulmonary artery was isolated and the pulmonary artery smooth muscle cells were isolated by using a 0.2% I-type collagenase digestion method. The proliferation of pulmonary artery smooth muscle cells was induced by the application of 20 ng/ ml of PDGF-BB. The proliferation and DNA synthesis of PASMCs were detected by CCK-8 and BRDU method at 12,24 and 48 hours with different concentrations of orange-skin intervention (12.5,25,50,100. mu.mol/ L). The cell cycle was analyzed by flow cytometry, and the cell cycle-related protein D1 and the cell cycle-related protein E were detected by flow cytometry. The expression of the cell cycle-related regulatory proteins, such as CDK2. CDK4, and the cell cycle kinase-inhibiting protein p27, was detected by the intervention of the PDGF-BB-stimulated pulmonary artery smooth muscle cells with 100. m u.mol/ L of orange peel, and Western Blotting was used to detect the total and phosphorylated ERK1/2, p38, JNK, AKT at 5,10,15 minutes after the effect. Glycogen synthase kinase 3 (GSK3). Results: The results of the detection of CCK-8 and BRDU showed that the orange peel can inhibit the proliferation of PDGF-BB and the synthesis of DNA, and the inhibition has time-dependent and concentration-dependent. The results of trypan blue staining showed that the survival rate of the treated PASMCs was not significantly toxic by the concentration groups (12.5,25,50,100. mu.mol/ L) in the experiment. The results of flow cytometry showed that 100. m u.mol/ L of orange peel was able to increase the number of cells in the G0/ G1 phase (62.0% to 0.6%-69.7% 0.3%). The cell cycle of S-phase cells decreased (25.7%-17.7%-1.2%), and the cell cycle was inhibited in G0/ G1-S phase. The results of Real Time PCR showed that the inhibitory effect of orange-skin on the cell cycle was related to the down-regulation of the expression of cyclin D1, cyclin E, CDK2, CDK4 and up-regulated P27. Further studies show that 100. m u.mol/ L of orange peel can inhibit the phosphorylation of AKT/ GSK3 and p38 signaling pathways without significant influence on ERK1/2 and JNK signaling pathways. Conclusion: The inhibitory effect of orange peel on the proliferation of PASMCs induced by PDGF-BB and the inhibitory effect of orange peel on the expression of the cell cycle-related protein D1, the cell cycle-related protein E, CDK2, CDK4 and the up-regulation of the cell cycle kinase inhibitory protein P27, and the cell cycle arrest in the G0/ G1-S phase, Further studies suggest that this inhibition is related to the inhibition of the level of phosphorylation of the AKT/ GSK3 and p38 signaling pathways. Orange skin has the potential to be a drug for clinical treatment of pulmonary hypertension.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1
本文编号：2441388
[Abstract]:Background: Pulmonary hypertension (PAH) is the mean pressure of the pulmonary artery in a quiet state of 25 mmHg. It is a clinical syndrome characterized by the continuous increase of pulmonary vascular resistance that can occur in various clinical diseases caused by different etiologies and pathogenesis. The study found that the pulmonary vessels in the pulmonary artery at high pressure could be changed as described below, such as intimal hyperplasia, media hypertrophy, adventitia proliferation, fibrosis, small vessel occlusion, thrombosis, inflammatory cell infiltration, and the like. In this, the abnormal proliferation of pulmonary artery smooth muscle cells (PASMCs) plays a key role in the reconstruction of pulmonary vessels. Long-term persistent pulmonary hypertension may eventually lead to right ventricular failure or even death, although this disease may have a serious effect on the level of health, but the common use of high pressure in the pulmonary artery can only improve the symptoms of the patient and not delay the development process. Therefore, it is of great value to find a substance capable of blocking and even reversing the abnormal proliferation of pulmonary artery smooth muscle cells, and to explore new therapeutic means and to improve the therapeutic effect and quality of life of patients with pulmonary hypertension. Objective: To establish a cell model of the proliferation of primary rat pulmonary artery smooth muscle cells induced by platelet-derived growth factor (PDGF)-BB and to study the effect of orange peel on the proliferation of pulmonary artery smooth muscle cells and its related mechanisms by using different concentrations of orange peel. To search for new drugs for the prevention and treatment of pulmonary vascular remodeling. Methods: SD rats weighing 150-200 g were selected and the pulmonary artery was isolated and the pulmonary artery smooth muscle cells were isolated by using a 0.2% I-type collagenase digestion method. The proliferation of pulmonary artery smooth muscle cells was induced by the application of 20 ng/ ml of PDGF-BB. The proliferation and DNA synthesis of PASMCs were detected by CCK-8 and BRDU method at 12,24 and 48 hours with different concentrations of orange-skin intervention (12.5,25,50,100. mu.mol/ L). The cell cycle was analyzed by flow cytometry, and the cell cycle-related protein D1 and the cell cycle-related protein E were detected by flow cytometry. The expression of the cell cycle-related regulatory proteins, such as CDK2. CDK4, and the cell cycle kinase-inhibiting protein p27, was detected by the intervention of the PDGF-BB-stimulated pulmonary artery smooth muscle cells with 100. m u.mol/ L of orange peel, and Western Blotting was used to detect the total and phosphorylated ERK1/2, p38, JNK, AKT at 5,10,15 minutes after the effect. Glycogen synthase kinase 3 (GSK3). Results: The results of the detection of CCK-8 and BRDU showed that the orange peel can inhibit the proliferation of PDGF-BB and the synthesis of DNA, and the inhibition has time-dependent and concentration-dependent. The results of trypan blue staining showed that the survival rate of the treated PASMCs was not significantly toxic by the concentration groups (12.5,25,50,100. mu.mol/ L) in the experiment. The results of flow cytometry showed that 100. m u.mol/ L of orange peel was able to increase the number of cells in the G0/ G1 phase (62.0% to 0.6%-69.7% 0.3%). The cell cycle of S-phase cells decreased (25.7%-17.7%-1.2%), and the cell cycle was inhibited in G0/ G1-S phase. The results of Real Time PCR showed that the inhibitory effect of orange-skin on the cell cycle was related to the down-regulation of the expression of cyclin D1, cyclin E, CDK2, CDK4 and up-regulated P27. Further studies show that 100. m u.mol/ L of orange peel can inhibit the phosphorylation of AKT/ GSK3 and p38 signaling pathways without significant influence on ERK1/2 and JNK signaling pathways. Conclusion: The inhibitory effect of orange peel on the proliferation of PASMCs induced by PDGF-BB and the inhibitory effect of orange peel on the expression of the cell cycle-related protein D1, the cell cycle-related protein E, CDK2, CDK4 and the up-regulation of the cell cycle kinase inhibitory protein P27, and the cell cycle arrest in the G0/ G1-S phase, Further studies suggest that this inhibition is related to the inhibition of the level of phosphorylation of the AKT/ GSK3 and p38 signaling pathways. Orange skin has the potential to be a drug for clinical treatment of pulmonary hypertension.
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R544.1
【参考文献】
相关期刊论文 前1条
1 吴媛媛;王贵佐;李满祥;;肺动脉平滑肌细胞增殖的分子信号机制研究进展[J];南方医科大学学报;2013年12期
，本文编号：2441388
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