【作者】 王春晓；

【导师】 夏伟；

【作者基本信息】 山东大学 ， 儿科学， 2013， 硕士

【摘要】 研究背景：钠氢交换体(NHE)是真核细胞内pH值的重要调节剂,到目前为止共发现了10种NHE亚型。其中NHE1主要表达在哺乳动物的心肌细胞、血管和肺,参与多种细胞功能,包括调节细胞内的pH值,稳定细胞容量,影响细胞的增殖和凋亡,调节细胞行为以及离子转运等。近年来,有大量文献表明,NHE1参与心肌损伤、重构、心肌肥厚及肺动脉高压的形成过程；更为重要的是,在动物实验中发现NHE1有促进细胞增殖的作用。左向右分流型先天性心脏病(congenital heart disease, CHD)的病理生理变化主要表现为肺循环血流量的显著增多。血管内皮细胞附着于血管管腔的内壁,因此,高肺血流可直接造成肺动脉内皮细胞(pulmonary arterial endothelial cells, PAECs)所受剪切力(shear stress, SS)的增加,由于内皮细胞的异质性,远端肺微血管内皮细胞更容易受到损伤,肺微血管内皮细胞损伤后激活、合成的多种细胞因子激发或是参与了肺动脉高压过程中肺血管的收缩和结构的重构。由于NHE1在慢性心衰及心肌肥厚等方面的研究较多,而在高肺血流所致肺动脉高压(pulmonary hypertension, PH)过程中的作用目前鲜有报道；同时关于肺动脉高压的发病机制中,当前研究的焦点更多的是有关肺动脉平滑肌的改变,从肺微血管内皮细胞(PMVECs)层次上的研究甚少。且多数实验研究局限于肺动脉高压慢性或中晚期的分子病理事件,因此本实验立项研究肺动脉高压发生的早期生物学效应。研究目的：研究流体剪切力对大鼠PMVECs NHE1表达的影响,探讨血流剪切力损伤肺微血管内皮细胞的早期作用机制。研究方法：培养大鼠肺微血管内皮细胞(PMVECs),而后应用多层流动腔装置对PMVECs进行流体冲击。依据剪切力作用的不同时间实验分为5组：正常对照组；剪切力作用15分钟组；剪切力作用30分钟组；剪切力作用45分钟组；剪切力作用60分钟组。实时荧光定量PCR检测NHE1的基因表达水平,Western-blot免疫印迹检测NHE1的蛋白表达。结果：在1.5*10-5N/cm2的剪切力作用下,实验组同对照组相比,NHE1的基因和蛋白水平表达明显上升(P<0.05),在升高的实验组中尤其以15分钟组最为明显,而后随着剪切力作用时间的延长,在作用30分钟和45分钟时NHE1基因和蛋白的表达出现了一个下降期,而在作用时间延长至60分钟时,NHE1的表达又显著增高。结论：血流剪切力上调NHE1在大鼠PMVECs中的表达,提示NHE1的表达上调可能损伤肺微血管内皮细胞,进而可触发肺动脉高压的发生。更多还原

【Abstract】 BACKGROUND:The Na+/H+exchanger (NHE) is one of the most studied plasma membrane mechanisms involved in proton transport. The10members of the NHE family described so far show a particular tissue distribution pattern. The isoform NHE-1is found in the plasma membrane of most mammalian cells and is normally described as the housekeeping isoform. It plays many different pHysiological roles in mammals, primarily responsible for the regulation of cellular pH, volume and growth. Recently many studies show that the role of NHE1in cardiac hypertropHy and chronic heart failure. Significant increasment of pulmonary blood flow is the main pathopHysiological change of left-right shunt congenital heart disease Because vascular endothelial cells attached to the inner wall of the vessel lumen, high pulmonary blood flow can lead to high shear force, causing pulmonary arterial endothelial cells (PAECs), especially pulmonary microvascular endothelial cell (PMVECs) to change its morpHology and function, secret some cytokines, which may play an important role in pulmonary vascular remodeling. The functional changes of PMVECs in the early stage of pulmonary vascular remodeling rarely have been reported. And the majority of experimental studies or animal experiments limited to chronic or late molecular pathological events.Objective:To explore the effect of Shear stress on Na+-H+exchanger isoform1in rat pulmonary microvascular endothelial cell, to discuss the early mechanism of injury on PMVECs by shear stress.Methods:Cultured rat PMVECs. Interfere PMVECs with the hydrodynamic system we designed, depending on the acting time, the PMVECs were randomly divided into control group,15minutes group、30minutes group、45minutes group、60minutes group, The gene and protein expression were determined by RT-PCR and Western blot, respectively.Results:When shear stress was1.5*10-5N/cm2, compared with the control group, the expression of NHE1at mRNA and protein levels was obviously increased in the groups acted on shear stress (P＜0.05), especially in the15minutes group. In the30minutes group and45minutes group, the NHE1expression was decreased compared with the15minutes group, but when the acting time was60minutes, the mRNA and protein levels of NHE1was significantly up-regulated.Conclusion:Fluid Shear stress can up-regulate the mRNA and protein lever of NHE1, which indicated that increased NHE1may has an action on early stage of pulmonary hypertension.更多还原