【作者】 黄海琼；

【导师】 姜桢；

【作者基本信息】 复旦大学 ， 麻醉学， 2009， 博士

【摘要】 肺动脉高压（Pulmonary Artery Hypertension,PAH）是指由多种病因所致肺血管床病变的基础上,肺循环阻力进行性增加,心导管检查平均肺动脉压静息状态下＞25 mmHg/或运动时＞30 mmHg,而肺毛细血管楔压≤15 mmHg的一类病理生理综合征。研究发现,肺毛细血管前阻力血管尤其是肺细小动脉的重塑是导致肺循环阻力和肺动脉压力进行性增加的主要因素,其主要病变之一是肺血管平滑肌细胞（Smooth Muscle Cell,SMC）增殖朋巴大、凋亡的失衡、细胞迁移以及细胞外基质合成、降解的异常,引起肺肌性细小动脉中膜肥厚、无肌细小动脉肌化,导致肺毛细血管前阻力血管管腔狭窄、闭塞,肺血管床面积减少,使肺循环阻力增加,从而引起肺动脉压持续增高和进行性恶化,最终致患者右心衰竭而死亡。因此,对人或实验动物肺血管SMC的分离和培养,是PAH发病机制研究中一种重要的体外细胞实验模型。内皮素-1（endothelin-1,ET-1）是新近研究较多的与肺血管重塑可能密切相关的一强缩血管多肽,其在PAH患者肺动脉中浓度显著升高,且与PAH的严重程度和预后密切相关,ET-1主要由肺血管内皮细胞合成,但肺血管SMC也具有合成和分泌ET-1的能力。在炎性因子的刺激下,血管SMC分泌ET-1的能力可上升100倍之多,达到血管内皮细胞的分泌水平。ET-1与其特异性的两个受体ET_AR和ET_BR结合,调节肺血管的收缩和舒张,此外,研究发现ET-1还可能诱导肺血管SMC增殖/月巴大、抗凋亡和向周围迁移,在PAH的发生和发展过程中可能发挥着重要的作用,因而我们推测PAH时异常升高的ET-1也许通过诱导肺细小动脉SMC增殖/月巴大与凋亡之间的失衡来参与肺细小动脉重塑的过程。为此,本课题拟在体外分离和培养大鼠肺细小动脉平滑肌细胞（RatPulmonary Arterial Microvascular Smooth Muscle Cells,RPMC）和大鼠肺大中动脉平滑肌细胞（Rat Pulmonary Large Arterial Smooth Muscle Cells,RPLC）,检测并比较两种细胞的增殖特性:在成功建立RPMC体外细胞培养模型的基础上,将ET-1基因瞬时转染RPMC,探讨过表达ET-1对RPMC增殖朋巴大和凋亡的影响及其可能信号转导机制。第一部分大鼠肺细小动脉和大动脉平滑肌细胞的分离培养及其增殖特性的比较目的:探讨体外分离和培养RPMC和RPLC的方法,并对两种细胞的生长和增殖特性进行比较。方法:SD大鼠麻醉后经右心室向肺动脉插管,依次灌注PBS、胰蛋白酶液、低融点琼脂糖和三氧化二铁混悬液,同时行气管插管并灌注低融点琼脂糖,夹闭左心房、肺动脉和气管,完整取下心肺组织置于预冷的DMEM不完全培养液中至琼脂糖凝固;沿胸膜下1 mm处剪下肺组织,在解剖显微镜下剔除动脉直径＞200μm的肺组织,剩余组织经剪切后放入Ⅳ型胶原酶中孵育;充分冲洗后再次剪切,用含20%胎牛血清的DMEM液混悬,并接种至预涂明胶的6孔板中培养。另取大鼠同上法麻醉后经右心室向肺动脉插管,依次灌注PBS、胰蛋白酶液,在解剖显微镜下分离大鼠肺大中动脉,Ⅳ型胶原酶消化外膜后,用组织块贴壁法培养。用细胞免疫荧光法和电镜进行培养细胞鉴定,用MTT法检测RPMC和RPLC细胞的增殖情况并绘制生长曲线,用流式细胞技术对两种细胞作细胞周期分析。结果:3-5 d可见RPMC自组织块周围爬出,而RPLC则在7-14 d左右爬出;RPMC和RPLC生长融合时均为长梭形,并呈培养的SMC特征性的“峰—谷”状生长方式;细胞免疫荧光法观察到SMC特征性的α-平滑肌肌动蛋白（α-smooth muscleactin,α-SMA）的表达;电镜也观察到其特征性的肌丝、密体和密斑;第3代培养细胞纯度均超过95%。MTT结果显示,RPMC的生长速度明显快于RPLC,在第4d时差异有统计学意义。流式细胞技术结果显示,与RPLC相比,RPMC中处于S期和G₂/M期的细胞比例增高,细胞增殖指数升高。结论:本实验为RPMC原代培养提供了一种切实可行的方法,并证实RPMC和RPLC的增殖特性之间存在差异,为体外实验研究存在于肺细小动脉中膜SMC病变的肺相关疾患,尤其是在对PAH发病机制的研究中,提供了更为直接、可靠的一种体外细胞研究模型。第二部分内皮素-1过表达对大鼠肺细小动脉平滑肌细胞凋亡的影响及可能作用机制目的:探讨过表达ET-1对体外培养的RPMC凋亡的影响,并进一步探讨其可能的作用机制。方法:三氧化二铁肺动脉灌注法体外分离和培养原代RPMC,重组真核表达质粒pMEXneo-ET1和pCDNA5-FRT-TO-ET1-3’UTR及其空载脂质体介导法瞬时转染RPMC并经实时荧光定量RT-PCR法和免疫印迹法鉴定后,用实时荧光定量RT-PCR法检测ET-1过表达对其受体ET_A和ET_B含量的影响,用流式细胞技术检测细胞凋亡的改变,并用免疫印迹法检测丝/苏氨酸蛋白激酶（Akt/PKB）的磷酸化水平和剪切的半胱天冬酶-3（Caspase-3）的水平变化。结果:瞬时转染的效率约为25%左右;瞬时转染RPMC 48h后,ET-1 mRNA表达分别为空载转染组的28.5倍和21倍,ET-1蛋白的表达分别为空载转染组的6.7倍和4.5倍;RPMC中ET_A受体mRNA表达水平明显高于ET_B受体。转染后RPMC中ET_A受体与ET_B mRNA表达水平均增高,但两者的比例保持不变:流式细胞技术结果显示,空载转染组RPMC凋亡的比例分别为10.99%和12.66%,转染组RPMC凋亡的比例为5.0%和4.1%,呈明显下降。免疫印迹法检测显示,过表达ET-1后,转染组RPMC的Akt/PKB的磷酸化水平升高,剪切的Caspase-3的表达水平下降。结论:ET-1可能经Akt/PKB-Caspase-3信号通路抑制RPMC的凋亡,从而在PAH的血管重塑过程中发挥着重要作用。第三部分内皮素-1过表达对大鼠肺细小动脉平滑肌细胞肥大特性的影响及可能信号转导机制目的:探讨过表达ET-1对体外培养的RPMC增殖/肥大的影响,并进一步探讨其可能的作用机制。方法:三氧化二铁肺动脉灌注法体外分离和培养原代RPMC,重组真核表达质粒pMEXneo-ET1和pCDNA5-FRT-TO-ET1-3’UTR及其空载脂质体介导法瞬时转染RPMC并经实时荧光定量RT-PCR法和免疫印迹法鉴定后,用流式细胞技术检测细胞周期和细胞体积的变化,用免疫印迹法和细胞免疫荧光法检测α-SMA的表达,同时检测细胞总蛋白/总DNA比的变化。并用免疫印迹法检测雷帕霉素靶蛋白（mTOR）和细胞外信号调节激酶（ERK1/2）的磷酸化水平。结果:与空载相比,转染48h后RPMC细胞周期分布和细胞增殖指数未见明显差异,但转染72h后检测到α-SMA合成功能增强,分别为空载转染组的1.3和1.2倍,细胞总蛋白/总DNA比也增高,分别为空载转染组的1.6和1.5倍。流式细胞术检测显示,与空载相比,转染72h后RPMC细胞体积增大,分别为空载转染组的1.1倍和1.2倍。免疫印迹法检测显示,转染48h后,Akt/PKB和mTOR的磷酸化水平升高,ERK1/2的磷酸化水平降低。结论:过表达ET-1可能经Akt/PKB-mTOR信号通路诱导RPMC肥大,从而可能在PAH的血管重塑过程中发挥着重要作用。更多还原

【Abstract】 IntroductionPulmonary arterial hypertension（PAH）,defined as a mean pulmonary artery pressure（mPAP） greater than 25 mmHg at rest or 30 mmHg with exercise and a pulmonary capillary wedge pressure less than 15 mmHg,as measured by right heart catheterization,is a multifactorial,serious and often progressive disorder that results in a sustained increase in pulmonary vascular resistance,right ventricular dysfunction and impairment in activity tolerance,and may lead to right-heart failure and death. The pathogenesis of PAH is complex and incompletely understood,but may share common histological abnormalities and pathophysiology.The irreversible morphologic changes in the pulmonary vascular bed lead to vascular remodeling including distal neomuscularization of the arterioles,intimal thickening,and medial hypertrophy.Abnormal collagen matrix is deposited within the adventitia later.Many of these changes can be attributed to the broken of the balance between hyperplasia/ hypertrophy and apoptosis of pulmonary arterial microvascular smooth muscle cells （SMC） and attention has therefore been focused on the related regulatory mechanisms of vascular SMC involved.Therefore,the successful isolating and culturing of the pulmonary arterial microvascular SMC in vitro represents the important model in the exploring of the pathogenesis of PAH.Endothelin-1（ET-1） is a potent 21 amino-acid peptide and mainly generated from the endothelium of blood vessels,as well as vascular SMC in part,and functions as a vasodilator and vasoconstrictor in a paracrine and autocrine manner on the ETA and ET_B receptors,which has been suggested to contribute to the local homeostasis of the pulmonary vasculature.Importantly,the amounts of ET-1 released by vascular SMC under inflammatory conditions are equivalent to those produced by the endothelium.Further,as the number of the vascular SMC in remodeling vessels is potentially greater than that of endothelial cells,the endogenous ET-1 production by these cells becomes increasingly important.In PAH patients,ET-1 is abnormally up-regulated in the circulation and pulmonary arteries,which is strongly correlated with measures of disease severity and survival in patients.Therefore,besides its vasoaction effects,ET-1 has been suggested to possess a direct mitogenic action, and/or apoptosis inhibition effect of vascular SMC in PAH.To investigate the effect of the hyperplasia,hypertrophy and apoptosis properties of the pulmonary arterial microvascular smooth muscle cell under the stimulating of the increasing ET-1 will pave a way to understand the pathophysiology of PAH,especially inducing some new point in preventing and treating strategies.In our current study,based on the successful culturing of rat pulmonary arterial microvascular smooth muscle cells（RPMC）,we investigated the effects of ET-1 overexpression on the hyperplasia,hypertrophy and apoptosis characteristics of RPMC and the possible signaling pathway involved was also explored through transiently transfecting RPMC with ET-1 gene.PartⅠMethod to cultivate rat pulmonary arterial microvascular and large arterial smooth muscle cells and comparison of their proliferative activityObjective:To define the method to cultivate RPMC and rat large arterial smooth muscle cells（RPLC） and to compare their proliferative activity.Methods:The pulmonary artery of an anesthetized adult rat was cannulated via the right ventricle.After orderly perfused with PBS,trypsin,low melting-point agarose and iron oxide particles mixture,the left atrium and pulmonary artery was clamped. Then the trachea was cannulated,and lung air space was filled with low melting-point argarose and the trachea was clamped.The whole lung and heart were isolated and transferred into the culture mediumⅠ[Dulbecco’s modified Eagle’s medium with penicillin（100 U/ml） and streptomycin（100μg/ml）]on ice to set the agarose.After subsequent trimming of the subpleural margin（no more than 1mm）,the lung was sliced and inspected with the microscope to ensure vessels of size less than 200μm. Following digested with collagenaseⅣ,rinsed with the culture mediumⅠ,the residue tissues were minced and suspended with culture mediumⅡ[20%fetal bovine serum （FBS） in the culture mediumⅠ]and then resuspended into the 6-well culture plate and incubated in humidified air with 5%CO₂ at 37℃.Meanwhile,same as the procedure, the first and second conduct artery were isolated after the trypsin perfusion finished with the microscope and dipped into collagenase to digest the adventitia.The aorta was clipped and the adventitia,the intima and the outer portion of the media of artery were mechanically scraped with the forceps.After repeated rinsed,the residuum was cut into approximately 5×5mm squares in culture plate and placed in an incubator under the same culture circumstance.The RPMC and RPLC were identified by immunofluorescence staining and electron microscope technology.Cell proliferation viability was tested by MTT assay.Flow cytometry was used to assess the cell cycle.Result:From 3 to 5 days,cells were visible migrating from the explants in RPMC, while from 7 to 10 days,cells were visible migrating from the explants in RPLC and at confluence,and both of the cells were elongated in shape and formed the typical "hill and valley" formation of cultured smooth muscle cells under the inverted contrast microscope.The cells were identified with the characteristicα-actin by the immunofluorescence and great amount of Intermediate filaments,dense plaques and dense bodies by electron microscope.The purity of the 3rd passage RPMC and RPLC was more than 95%.Compared with RPLC,RPMC exhibited a faster growth rate in 4d with MTT and higher cell Proliferate Index with Flow cytometry.Conclusion:The method to cultivate RPMC via tissue perfusion is feasible and there is a different proliferative characteristic between RPMC and RPLC.Further researches using this cell model may pave a way to explore the pathogenesis of the lung diseases with pulmonary arterial microvascular smooth muscle cell disorders,e.g. pulmonary hypertension. PartⅡThe effect and mechanism of overexpression of endothelin-1 on apoptosis of rat pulmonary arterial microvascular smooth muscle cells in vitroObjective:To investigate the effect and mechanism of overexpression of ET-1 on the apoptosis of RPMC in vitro.Methods:The RPMC was obtained and cultured by the lung tissue perfusion method, then the third generation RPMC was transient transfected with the pMEXneo-ET1 and pCDNA5-FRT-TO-ET1-3’UTR plasmids as well as the empty vector respectively via Lipofectamine^TM 2000.Real time RT-PCR was used to assess the ET_A and ET_B levels and flow cytometry was used to assess the apoptosis of RPMC.Akt and Caspase-3 were detected by Western blot assay.Results:The transfection efficiency is about 25%.The mRNA of ET_A receptor was higher than that of ET_B receptor.Both of the ET_A receptor and the ET_B receptor mRNA in the transfected RPMC was increased,however,the ratio between the ET_A receptor and the ET_B receptor mRNA was the same as the control.Flow cytometry analysis revealed that the apoptosis was decreased after RPMC was transfected with the ET-1 for 48 h.The Western blotting results showed that overepression of ET-1 in RPMC increased the phosphorylation of Akt and reduced the Cleaved Caspase-3.Conclusions:Overexpression of the ET-1 inhibits the apoptosis of RPMC and activation of Akt/PKB-Caspase-3 signaling pathway may be involved in its mechanism,which may play a role in the remodeling of the pulmonary microvascular arteries.PartⅢThe effect and mechanism of overexpression of endothelin-1 on hypertrophy of rat pulmonary arterial microvascular smooth muscle cells in vitroObjective:To investigate the effects and mechanism of overexpression of ET-1 on the hypertrophy of RPMC in vitro.Methods:Lung tissue perfusion method was used to obtain the primary RPMC and it was identified via immunofluorescence staining and electron microscope technique. The RPMC was transient transfected with the pMEXneo-ET1 and pCDNA5-FRT-TO-ET1-3’UTR plasmids as well as the empty vector respectively via Lipofectamine^TM 2000.Flow cytometry was used to assess the cell cycle and cell size of RPMC,as well as the ratio of the protein/DNA of the transfected RPMC on the 72 h.Akt and mTOR were detected by Western blot assay to investigate the molecular mechanism.Results:Primary RPMC was obtained successfully,and the mRNA of ET-1 was higher than that of the empty vectors.Flow cytometry analysis revealed that the cell size was increased after RPMC was transfected with the ET-1 for 72 h.The ratios of the protein/DNA of the transfected RPMC were extended than the empty vectors on 72 h.The Western blotting results showed that overepression of ET-1 in RPMC increased the phosphorylation of Akt and mTOR.Conclusions:Overexpression of the ET-1 induces the hypertrophy of RPMC and activation of Akt/PKB-mTOR signaling pathway may be involved in its mechanism, which may be responsible for the remodeling of the pulmonary microvascular arteries.更多还原