肺动脉高压的实验与临床研究

【作者】 张洪亮；

【导师】 柳志红； 熊长明； 倪新海；

【作者基本信息】 中国协和医科大学 ， 内科学， 2010， 博士

【摘要】 背景肺动脉高压是一处理极为棘手,高致死率和致残率的病理生理综合征,被认为是“假恶性”肿瘤。肺动脉高压与细胞内膜运输障碍有关。本研究旨在探讨膜运输相关蛋白在肺动脉高压发病过程中的作用。方法选用Sprague-Dawley大鼠经腹腔注射野百合碱建立肺动脉高压动物模型；用吡咯野百合碱处理人肺动脉内皮细胞建立肺动脉高压细胞模型；并在人肺动脉内皮细胞中采用Nem干预Nsf蛋白的活性。采用反转录——实时定量PCR和Western杂交技术检测了膜运输相关蛋白Nsf,α-Snap、Snap23和Syntaxin4以及内皮型一氧化氮合酶、小凹蛋白1和骨形成蛋白受体2在动物模型和细胞模型中的mRNA和蛋白表达变化。Western杂交检测凋亡相关蛋白在肺动脉高压动物模型、细胞模型和NEM干预后细胞内的表达变化,从而检测细胞凋亡的情况。结果在体内实验水平,与同步对照相比,实验组大鼠腹腔注射野百合碱后,Nsf,α-Snap、Snap23和Syntaxin4的mRNA表达呈先增高后降低的趋势。Nsf、α-Snap和Snap23在刺激1天后显著增高,α-Snap从刺激后7天开始下降,Nsf和Snap23于14天开始下降,表达量极少。Syntaxin4的mRNA表达在刺激2天后显著增高,14天和21天后基本检测不到。Western杂交显示随着实验动物肺动脉高压的形成,Nsf、α-Snap、SNAP23和Syntaxin4的蛋白表达亦出现先显著增高,后明显降低,甚至检测不到表达的变化趋势,与mRNA的表达变化趋势基本一致。内皮型一氧化氮合酶于大鼠腹腔注射野百合碱7天后mRNA表达显著增高,14天后下降,21天时基本检测不到。小凹蛋白1和骨形成蛋白受体2的表达从14天开始显著减少,21天时检测到极少量表达；Western杂交显示内皮型一氧化氮合酶、小凹蛋白1和骨形成蛋白受体2在野百合碱刺激14天后显著减少。在体外实验水平,α-Snap和Snap23的mRNA表达在吡咯野百合碱刺激4-8h内无明显变化,12-72h之间增加；Nsf、Syntaxin4和小凹蛋白1的mRNA表达与对照相比无显著变化；而内皮型一氧化氮合酶的mRNA表达在吡咯野百合碱刺激24-72h内显著增加；骨形成蛋白受体2的mRNA表达早期无显著变化,12-24h后显著增加,48h后降至正常水平。Western杂交检测结果显示Nsf、内皮型一氧化氮合酶和小凹蛋白1在吡咯野百合碱刺激后期减少；α-Snap从12h开始轻度增加。骨形成蛋白受体2先增加,后降低,甚至检测不到表达。用NEM处理人肺动脉内皮细胞后,Nsf的mRNA表达随刺激时间延长显著增高达40倍以上；α-Snap、Snap23和Syntaxin4的mRNA表达亦显著增高2-16倍。内皮型一氧化氮合酶和小凹蛋白1的mRNA表达呈先增高后降低的趋势。骨形成蛋白受体2的表达显著增高。Western杂交检测结果显示,与对照相比,Nsf、α-Snap、Syntaxin4和内皮型一氧化氮合酶的蛋白表达呈逐渐降低的趋势；Snap23的蛋白表达随Nem处理时间的延长先增高,后降低；小凹蛋白1的表达无明显变化；骨形成蛋白受体2蛋白表达在NEM处理48h后增加。在肺动脉高压的动物模型、细胞模型和用NEM处理后的人肺动脉内皮细胞中,Western杂交检测显示,procaspase-3表达下降,Fas或Bax表达增加,提示细胞凋亡增加。结论膜运输相关蛋白和内皮型一氧化氮合酶、小凹蛋白1以及骨形成蛋白受体2参与了肺动脉高压的病理生理过程。膜运输相关蛋白的表达水平在肺动脉高压形成过程中最先发生变化,膜运输障碍机制在肺动脉高压的发生和发展过程中发挥了重要作用。更多还原

【Abstract】 Background Pulmonary hypertension is a pathophysiologic syndrome, which is difficult to handle and with high mortality and disability. It is considered a pseudo-malignant tumor. Pulmonary hypertension is associated with intracellular membrane trafficking. The aim of this study is to investigate the role of membrane trafficing associated proteins in the pathogenesis of pulmonary hypertension.Methods Sprague-Dawley rats were used to make the animal model of pulmonary hypertension by intraperitoneal injection of monocrotaline. The cellular model of pulmonary hypertension was made with human pulmonary arterial endothelial cells pretreated with monocrotaline pyrrole. And Nem was used to inhibit Nsf in human pulmonary arterial endothelial cells. The mRNA and protein level of membrane trafficking associated proteins (Nsf, a-Snap, Snap23 and Syntaxin4) and endothelial nitric oxide synthase, caveolin-1 and bone morphogenic protein receptorⅡin animal model and cellular model were tested by reverse transcription-real time PCR and Western Blot. Also the apoptotic proteins were tested by Western Blot.Results In rat, the mRNA levels of Nsf, a-Snap, Snap23 and Syntaxin4 in monocrotaline group were first increased, then decreased. The mRNA expression of Nsf, a-Snap and Snap23 were enhanced in 1 day after injection of monocrotaline. Then a-Snap started to decrease in the 7th day, and Nsf and Snap23 in the 14th day, to a very low level. The mRNA expression of syntaxin4 was elevated in 2 days after injection of monocrotaline, and down-regulated from 14 days to a level hardly tested. The changes of protein levels of Nsf, a-Snap, Snap23 and Syntaxin4 were similar to changes of mRNA level. The mRNA level of endothelial nitric oxide synthase was increased in the 7th day, decreased from the 14th day, and can not be detected in the 21th day. While caveolin-1 and bone morphogenic protein receptor II also decreased from the 14th day to a very low level in the 21th day. Western blot showed that endothelial nitric oxide synthase, caveolin-1 and bone morphogenic protein receptor II decreased from the 14th day.In monocrotaline pyrrole treated cells, the mRNA of a-Snap and Snap23 started to increase from 12 hours after treatment with monocrotaline pyrrole, and endothelial nitric oxide synthase from the 24th hour. The mRNA level of bone morphogenic protein receptor II strated to elevate from the 12th hour, then decreased to normal level from the 48th hour. And there was no change in the mRNA of Nsf, Syntaxin4 and caveolin-1. Western blot showed that Nsf, endothelial nitric oxide synthase and caveolin-1 decreased the the late phase. The protein changes of a-Snap and bone morphogenic protein receptor II were similar to the corresponding mRNA.After pretreatment of human pulmonary arterial endothelial cells with NEM, the mRNA levels of Nsf, a-Snap, Snap23, Syntaxin4 and bone morphogenic protein receptor II were increased greatly. The mRNA levels of endothelial nitric oxide synthase and caveolin-1 were first increased, then decreased. Western blot showed that Nsf, a-Snap, Syntaxin4 and endothelial nitric oxide synthase gradually decreased; Snap23 was first increased, then decreased; bone morphogenic protein receptor II increased from the 48th hour; no change in caveolin-1.In animal and cellular models and NEM treated cells, western blot showed the level of procaspase-3 was decreased and the level of Fas or Bax was elevated, which meant cellular apoptosis was enhanced.Conclusions Membrane trafficking associated proteins and endothelial nitric oxide synthase, caveolin-1 and bone morphogenic protein receptor II were involved in pathogenesis of pulmonary hypertension. The expression of membrane trafficking associated proteins was firstly changed in pummonary hypertension. Membrane trafficking dysfunction plays an important role in the pathogesis and progression of pulmonary hypertension.更多还原