肺动脉高压中TGF-β1和PPAR-γ调控ET-1表达的分子机制研究

发布时间：2018-02-21 10:32

本文关键词： 肺动脉高压 ET-1 TGF-β1 PPAR-γ 信号转导　出处：《暨南大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景:肺动脉高压(Pulmonary hypertension,PH)是指肺血管阻力持续增高超过一定界值的病理生理状态,可导致右心衰竭而危及生命。目前普遍认为PH的病理生理机制主要涉及血管收缩、细胞增殖、血管重塑、血栓形成和血管内皮损伤,多种因素的综合作用使肺血管压力进行性升高,最终导致右心功能衰竭和死亡,临床治疗效果不佳,发病机制不明。ET-1与肺血管重塑及肺动脉压力增高密切相关,然而具体的分子机制仍不清楚。已有研究表明ET-1、TGF-β1及PPAR-γ均参与了PH的病理发生及发展过程,PPAR-γ信号转导、MAPK及Smad信号通道激活以及ET-1高表达可能是肺动脉高压的病理生理基础,其具体机制及信号转导通路至今尚未完全明确,值得进一步研究。目的:通过检测ET-1、TGF-β1在PH患者血液中的表达水平、PPAR-γ在PH患者肺组织中的表达水平、TGF-β1对A549细胞表达ET-1的影响及其细胞信号转导机制、PPAR-γ对TGF-β1刺激A549细胞表达ET-1的影响及其细胞信号转导机制等研究,以阐明在A549细胞中TGF-β1及PPAR-γ对ET-1表达调控的分子机制。方法:第一部分:根据患者病史、临床症状及超声心动图结果选取47例PH患者,采用ELISA检测患者血清中ET-1及TGF-β1蛋白的表达水平;选取PH患者肺组织8例,通过免疫组织化学染色法研究PPAR-γ在PH患者肺组织的定位和表达及其临床意义。第二部分:根据临床研究的结果,在细胞水平上,研究TGF-β1、PPAR-γ对ET-1表达的影响,深入研究其分子调控机制。1.选择A549细胞,用TGF-β1刺激A549细胞后,通过q RT-PCR和ELISA检测ET-1的m RNA及蛋白表达水平,通过Western blot检测MAPK(磷酸化p38)、JNK/SAPK和NFκB(磷酸化p65,p50亚基)等信号通道的激活情况,结合使用p38激酶抑制剂SB203580等信号通道选择性抑制剂研究参与ET-1释放的主要信号转导途径。2.采用RNA干扰技术沉默PPAR-γ基因并转染A549细胞,另用PPAR-γ的抑制剂S2871作用于A549细胞,通过ELISA和q RT-PCR检测沉默了PPAR-γ基因及加入抑制剂S2871后的A549细胞ET-1蛋白和m RNA表达水平有何差异;构建含PPAR-γ基因的质粒并转染A549细胞,另用PPAR-γ的激动剂S2505作用于A549细胞,通过ELISA和q RT-PCR检测其在TGF-β1刺激作用下的ET-1分泌情况;采用Western blot检测研究PPAR-γ在TGF-β1刺激A549细胞释放ET-1这一过程中对其可能的细胞信号传导通路所起的作用。结果:第一部分:PH组患者血液中ET-1和TGF-β1蛋白表达水平较正常对照组明显增高,ET-1及TGF-β1蛋白的表达水平与肺动脉收缩压之间存在相关性,且增高的ET-1和TGF-β1之间也存在相关性;PPAR-γ在肺组织中主要表达于肺泡上皮细胞和血管内皮细胞的胞核中,PH患者肺组织的PPAR-γ表达较正常对照组明显减少。第二部分:1.在体外培养的A549细胞中,q RT-PCR和ELISA结果提示TGF-β1可诱导ET-1的m RNA及蛋白表达增加,同时WB检测到phospho-p38 MAPK及phospho-Smad2表达增加,p38的特异性抑制剂SB203580可抑制TGF-β1诱导的ET-1 m RNA及蛋白表达,提示TGF-β1通过p38 MAPK及Smad2信号通路诱导ET-1的表达增加。2.PPAR-γ沉默或用PPAR-γ抑制剂S2871预处理A549细胞,q RT-PCR和ELISA结果显示TGF-β1诱导的ET-1 m RNA及蛋白表达均增加,WB同时检测到phospho-p38 MAPK及phospho-Smad2表达增加;PPAR-γ超表达或用PPAR-γ激动剂S2505预处理A549细胞,q RT-PCR和ELISA结果显示TGF-β1诱导的ET-1 m RNA及蛋白表达均下降,WB同时检测到phospho-p38 MAPK及phospho-Smad2表达降低,提示PPAR-γ可通过p38 MAPK及Smad2调控TGF-β1诱导的ET-1表达。结论:PH患者血液中的ET-1、TGF-β1表达增加,随着PH严重程度的加重,ET-1及TGF-β1蛋白的表达水平越高,且增高的ET-1和TGF-β1之间存在相关性;PH患者肺组织中的PPAR-γ表达减少;在体外培养的A549细胞中,TGF-β1通过p38 MAPK及Smad2信号通路诱导ET-1分泌增加且这一过程受PPAR-γ调控。
[Abstract]:Background: pulmonary arterial hypertension (Pulmonary, hypertension, PH) refers to the pulmonary vascular resistance increased more than a certain critical value of pathological and physiological conditions, can lead to right heart failure and life-threatening. It is generally believed that the pathophysiological mechanism of PH vasoconstriction, cell proliferation, vascular remodeling, thrombosis and vascular endothelial injury, comprehensive a variety of factors make the pulmonary vascular pressure increased, eventually leading to right heart failure and death, clinical treatment effect, unknown pathogenesis.ET-1 and pulmonary vascular remodeling and pulmonary artery pressure closely related, but the detailed mechanism is still unclear. Studies have shown that ET-1, TGF- and PPAR- were gamma beta 1 involved in the pathology of PH occurrence and development process of PPAR- signal transduction, MAPK and Smad signal pathway activation and high expression of ET-1 may be the pathophysiological basis of pulmonary hypertension and its mechanism And the signal transduction pathway is still not completely clear, it is worthy of further study. Objective: to detect ET-1, TGF- beta 1 in blood of patients with PH expression levels, the expression level of PPAR- gamma in lung tissue in patients with PH, ET-1 and its effect mechanism of signal transduction of TGF- beta 1 expression in A549 cells, the expression of ET-1 effect and the signal transduction mechanism of PPAR- gamma beta 1 TGF- stimulation of A549 cells, A549 cells in order to elucidate the TGF- beta 1 and PPAR- gamma on the expression of ET-1 molecular mechanism. Methods: the first part: according to the patient history, clinical symptoms and echocardiographic findings from 47 cases of PH patients, the expression level of serum in patients with ELISA detection of ET-1 and TGF- protein beta 1; a total of 8 cases of lung tissue in patients with PH by immunohistochemical staining method of PPAR- gamma in lung tissue of patients with PH localization and expression and its clinical significance. The second part: according to the clinical research The results, at the cellular level, TGF- beta 1, PPAR- gamma on the expression of ET-1, in-depth study of the molecular mechanism of.1. A549 cells with TGF- beta 1 after A549 cells were stimulated by m, the expression level of RNA protein and Q RT-PCR and ELISA ET-1 detection, blot detection of MAPK by Western (phosphorylated p38), JNK/SAPK and NF K B (phosphorylated p65, subunit P50) activation of signal channel, signal transduction binding studies using inhibitors of p38 kinase SB203580 signal channel selective inhibitors involved in ET-1 release pathway of.2. by RNA interference silencing PPAR- gene and transfected into A549 cells, another inhibitor S2871 PPAR- gamma in A549 cells, ELISA and Q by RT-PCR detection of silent PPAR- gene and A549 were added ET-1 protein and m RNA inhibitor S2871 expression level difference; construct containing PPAR- gamma gene plasmid and transfected into A549 cells, the other With a PPAR- gamma agonist S2505 in A549 cells by ELISA and Q RT-PCR detected in the TGF- beta 1 stimulation of ET-1 secretion by Western blot; detection of PPAR- gamma release ET-1 this process in the possible cellular signaling pathways play a role in beta 1 TGF- stimulation of A549 cells. Results: the first part: the expression of PH ET-1 and TGF- beta 1 protein levels in the blood of patients were significantly higher than those in control group, the expression level of ET-1 and TGF- beta 1 protein and pulmonary artery systolic pressure and the correlation between increased ET-1 and TGF- beta correlation also existed between 1; PPAR- gamma in lung tissue expression in alveolar epithelial cells and vascular endothelial cells in the nucleus, the expression of PPAR- gamma lung tissues of PH patients compared with normal control group decreased significantly. The second part: 1. in vitro cultured A549 cells, Q RT-PCR and ELISA results suggest that TGF- beta 1 can induce ET-1 M RNA and protein expression increased, while WB detected phospho-p38 MAPK and increase the expression of phospho-Smad2, a specific inhibitor of SB203580 p38 can inhibit the expression of TGF- 1 induced ET-1 m RNA and protein expression of TGF- beta 1 by p38 MAPK and Smad2 signal pathway induced by ET-1 increased.2.PPAR- or PPAR- silencing gamma gamma inhibitor S2871 pre treatment of A549 cells, Q RT-PCR and ELISA showed that the expression of TGF- beta 1 induced ET-1 m and protein of RNA were increased, WB and phospho-p38 detected MAPK and phospho-Smad2 expression increased; PPAR- overexpression or PPAR- gamma agonist S2505 pretreatment of A549 cells with Q, RT-PCR and ELISA showed that the expression of TGF- beta 1 induced by ET-1 m RNA and protein were decreased, WB and phospho-p38 detected MAPK and phospho-Smad2 expression decreased, suggesting that PPAR- through p38 MAPK and Smad2 gamma beta 1 regulates TGF- induced ET-1 expression. Conclusion: in the blood of patients with PH ET-1, TGF- beta 1 expression increased with increasing severity of PH, the expression level of ET-1 and TGF- beta 1 protein is higher, and the increase of ET-1 and TGF- beta correlation exists between 1; PPAR- gamma PH in lung tissue in patients with decreased expression; A549 cells cultured in vitro, TGF- beta 1 by p38 MAPK and Smad2 signaling pathway induced by increased secretion of ET-1 and this process by PPAR- gamma regulation.

【学位授予单位】：暨南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R544.1

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