橙皮素對(duì)血小板源性生長(zhǎng)因子-BB所致肺動(dòng)脈平滑肌細(xì)胞增殖的作用研究
發(fā)布時(shí)間：2019-03-16 12:47
【摘要】：背景：肺動(dòng)脈高壓(Pulmonary arterial hypertension, PAH)是指處于安靜狀態(tài)下肺動(dòng)脈平均壓25mmHg。它是由不同病因和發(fā)病機(jī)制引起的在多種臨床疾病中均可出現(xiàn)的以肺血管阻力持續(xù)升高為特點(diǎn)的臨床綜合征。研究認(rèn)為,肺動(dòng)脈高壓時(shí)肺血管可以出現(xiàn)下述諸如內(nèi)膜增生,中膜肥厚,外膜增殖、纖維變性,小血管閉塞,血栓形成,炎癥細(xì)胞浸潤(rùn)等等改變。在這之中,肺動(dòng)脈平滑肌細(xì)胞(pulmonary aterial smooth muscle cells, PASMCs)的異常過(guò)度的增殖在肺血管的重構(gòu)中起到關(guān)鍵作用。長(zhǎng)期持續(xù)的肺動(dòng)脈高壓最終會(huì)導(dǎo)致右心室衰竭甚至死亡,盡管本病對(duì)健康水平可造成嚴(yán)重影響,但目前肺動(dòng)脈高壓的常用藥物均只能改善患者的癥狀而不能延緩其發(fā)展進(jìn)程。因此尋找能夠阻滯甚至逆轉(zhuǎn)肺動(dòng)脈平滑肌細(xì)胞異常增殖的物質(zhì),探索新的治療手段,對(duì)提高肺動(dòng)脈高壓患者的治療效果和生活質(zhì)量具有重要的價(jià)值。目的：建立血小板源性生長(zhǎng)因子(platelet-derived growth factor, PDGF)-BB誘導(dǎo)的原代大鼠肺動(dòng)脈平滑肌細(xì)胞的增殖細(xì)胞模型,并通過(guò)使用不同濃度的橙皮素進(jìn)行干預(yù),探討橙皮素對(duì)肺動(dòng)脈平滑肌細(xì)胞增殖的影響及其相關(guān)機(jī)制,為肺血管重構(gòu)的防治尋找新藥物。方法：選擇體重處于150-200 g的SD大鼠,分離肺動(dòng)脈,選用0.2%I型膠原酶消化法分離肺動(dòng)脈平滑肌細(xì)胞。應(yīng)用20ng/ml PDGF-BB誘導(dǎo)肺動(dòng)脈平滑肌細(xì)胞增殖創(chuàng)建細(xì)胞模型。應(yīng)用不同濃度橙皮素干預(yù)(12.5、25、50、100μmol/L),通過(guò)CCK-8及BRDU法于12、24、48小時(shí)檢測(cè)PASMCs的增殖和DNA合成；不同濃度橙皮素(12.5、25、50、100μmol/L)處理PASMCs于12、24、48小時(shí)后通過(guò)0.4%濃度的臺(tái)盼藍(lán)染色法檢測(cè)細(xì)胞存活率；予100μmol/L橙皮素干預(yù)PDGF-BB刺激肺動(dòng)脈平滑肌細(xì)胞24小時(shí)后,采用流式細(xì)胞儀分析細(xì)胞周期,實(shí)時(shí)定量PCR (RT-PCR)檢測(cè)細(xì)胞周期相關(guān)性蛋白D1,細(xì)胞周期相關(guān)性蛋白E,CDK2.CDK4和細(xì)胞周期激酶抑制蛋白p27等細(xì)胞周期相關(guān)調(diào)控蛋白mRNA的表達(dá)；用100μmol/L橙皮素干預(yù)PDGF-BB刺激的肺動(dòng)脈平滑肌細(xì)胞,于作用后5、10、15分鐘應(yīng)用Western Blotting的方法來(lái)檢測(cè)比較總的以及磷酸化的ERK1/2,p38, JNK, AKT,糖原合酶激酶3β(GSK3β)。結(jié)果：CCK-8和BRDU法檢測(cè)結(jié)果表明橙皮素能夠抑制PDGF-BB刺激的PASMCs增殖及DNA的合成,并且這種抑制作用具有時(shí)間依賴性和濃度依賴性。臺(tái)盼藍(lán)染色的結(jié)果表明本實(shí)驗(yàn)應(yīng)用的各濃度組(12.5,25,50, 100μmol/L)橙皮素對(duì)處理過(guò)后的PASMCs其存活率沒(méi)有顯著的毒性作用；流式細(xì)胞儀分析結(jié)果表明100μmol/L橙皮素能夠使G0/G1期細(xì)胞增多(62.0±0.6%-69.7±0.3%),S期細(xì)胞減少(25.7±0.9%-17.7±1.2%),抑制細(xì)胞周期于G0/G1-S期,Real Time PCR結(jié)果表明橙皮素對(duì)細(xì)胞周期的抑制作用與下調(diào)細(xì)胞周期蛋白D1,細(xì)胞周期蛋白E,CDK2, CDK4和上調(diào)P27的mRNA表達(dá)有關(guān)；進(jìn)一步研究表明100μmol/L橙皮素能夠抑制AKT/GSK3β和p38信號(hào)通路的磷酸化,而對(duì)ERK1/2和JNK信號(hào)通路無(wú)明顯影響。結(jié)論：橙皮素可以抑制PDGF-BB誘導(dǎo)的PASMCs增殖,橙皮素的抑制作用是通過(guò)下調(diào)細(xì)胞周期相關(guān)性蛋白D1,細(xì)胞周期相關(guān)性蛋白E,CDK2,CDK4及上調(diào)細(xì)胞周期激酶抑制性蛋白P27的mRNA表達(dá),將細(xì)胞周期阻滯于G0/G1-S期來(lái)實(shí)現(xiàn),進(jìn)一步研究提示這種抑制作用與抑制AKT/GSK3β和p38信號(hào)通路的磷酸化水平有關(guān)。橙皮素有潛力成為臨床治療肺動(dòng)脈高壓的藥物。
[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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R544.1
本文編號(hào)：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.
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R544.1
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 吳媛媛;王貴佐;李滿祥;;肺動(dòng)脈平滑肌細(xì)胞增殖的分子信號(hào)機(jī)制研究進(jìn)展[J];南方醫(yī)科大學(xué)學(xué)報(bào);2013年12期
，本文編號(hào)：2441388
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