【作者】 周建国；

【导师】 肖颖彬；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 背景与目的:先天性心脏病合并肺动脉高压的发病机制复杂,参与因子众多,其发病机理至今尚未完全清楚。肾素-血管紧张素系统(RAS)在维持心血管功能稳定、水电解质平衡、细胞生长、组织纤维化等方面起着十分重要的作用,尤其对动脉血压的调节有重要意义。血管紧张素Ⅱ(AngiotensinⅡ,AngⅡ)是RAS的主要成分,它的生理作用主要由AngⅡ-1型受体(AT1)介导。AngⅡ与AT1在体循环中的作用已较清楚,但对其在肺动脉高压肺血管重塑的发生发展过程中作用的相关研究,文献报道很少。水通道(AQPs)是一组与水通透有关的细胞膜转运蛋白。AQP5分布于气管、主支气管直至细支气管的柱状上皮细胞顶膜、粘膜下腺腺泡细胞顶膜及I型肺泡上皮细胞,与与呼吸道上皮的屏障功能、细胞膜通透性以及呼吸道水平衡功能有关。本研究旨在通过检测AngⅡ信号通路及AQP5在先天性心脏病合并肺动脉高压患者肺组织中的变化,初步探讨其意义,以期为先天性心脏病合并肺动脉高压的防治提供新的思路和治疗靶点。方法:1.本研究符合医学道德伦理规范。选取拟手术治疗的室间隔缺损患者40例,根据术前超声多普勒测定的平均肺动脉压(mPAP),将患者分为4组。即无肺动脉高压组(组1,mPAP≤25mmHg,n=10);轻度肺动脉高压组(组2A,mPAP26mmHg～35mmHg,n=10);中度肺动脉高压组(组2B,mPAP36mmHg～45mmHg,n=10);重度肺动脉高压组(组2C,mPAP≥46mmHg,n=10)。2.各组取术前动脉血,以放射免疫法检测血浆血管紧张素Ⅱ含量。3.各组术中取肺组织,以免疫组化法检测肺组织血管紧张素Ⅱ1型受体表达、以Western blot检测肺组织磷酸化信号转换和转录激活因子1表达、以EMSA检测肺组织核转录因子-κB的活化水平、以RT-PCR检测肺组织水通道蛋白5 mRNA的表达、以Western blot检测肺组织水通道蛋白5的表达。4.在上述实验结果的基础上,另将8例先天性心脏病室间隔缺损合并重度肺动脉高压患者,观察血管紧张素转换酶抑制剂Captopril对先心病重度肺动脉高压患者肺组织AngⅡ信号通路及AQP5表达的影响。结果:1.随着肺动脉压的升高,患者血浆血管紧张素Ⅱ水平均增高,肺动脉高压组(组2A、组2B、组2C)均明显高于无肺动脉高压组(组1)(P<0.05)。2.随着肺动脉压的升高,患者肺组织血管紧张素Ⅱ1型受体表达均增高,肺动脉高压组(组2A、组2B、组2C)均明显高于无肺动脉高压组(组1)(P<0.05);磷酸化STAT1表达均增高,肺动脉高压组(组2A、组2B、组2C)均明显高于无肺动脉高压组(组1)(P<0.05);核转录因子-κB活化程度降低,肺动脉高压组(组2A、组2B、组2C)均明显低于无肺动脉高压组(组1)(P<0.05);AQP5 mRNA表达降低,肺动脉高压组(组2A、组2B、组2C)均明显低于无肺动脉高压组(组1)(P<0.05);AQP5蛋白含量降低;肺动脉高压组(组2A、组2B、组2C)均明显低于无肺动脉高压组(组1)(P<0.05)。3.血管紧张素转换酶抑制剂Captopril能抑制重度肺动脉高压患者肺组织AngⅡ信号通路的活性,并增加AQP5的表达,统计分析显示各组间均有显著性差异(P <0.05)。结论:1.在先天性心脏病肺动脉高压患者中,AngⅡ信号通路发生了明显的变化。随着肺动脉高压的升高,血浆AngⅡ浓度升高;肺组织AT1和磷酸化STAT1含量增高,核转录因子-κB活性降低,提示AngⅡ信号通路表达上调在继发性肺动脉高压发生发展过程中起重要作用。2.随着肺动脉压的升高,先天性心脏病患者肺组织中AQP5 mRNA和AQP5蛋白表达均降低,提示肺泡上皮细胞水平衡能力降低,可能是先天性心脏病合并肺动脉高压患者术后易发生肺水肿的重要机制。3.在重度肺动脉高压患者,术前口服血管紧张素转换酶抑制剂Captopril治疗,可以抑制肺组织血管紧张素Ⅱ信号通路活性,表现为血浆AngⅡ浓度降低;肺组织AT1和磷酸化STAT1含量减少,核转录因子-κB活性增强;而肺组织AQP5mRNA和AQP5蛋白表达相应增加。这提示,针对血管紧张素Ⅱ信号通路与水通道蛋白5的治疗,有助于改善肺组织血管重构和肺泡上皮水平衡能力。更多还原

【Abstract】 Background and ObjectThe pathogenesis of pulmonary hypertension with congenital heart disease has not been elucidated completely due to its complexity and numerous factors involved. Renin-angiotensin system (RAS) plays an important role in maintaining cardiovascular function stable, water-electrolyte balance, cell growth, and fibrous degeneration of tissues. Furthermore, it was of great importance to regulate pulmonary circulation. Angiotensin II (Ang II) was the main ingredient of RAS. Its physio-function was mainly mediated through angiotensin II receptor type 1 (AT1). The role played by Ang II and AT1 in general circulation has been detected clearly, and a consensus concerning its vital function in the process of myocardial apoptosis and remodeling has been achieved. However, few studies on its role in the genesis and development of hypertensive pulmonary vascular remodeling have been reported. Aquaporin (AQPs) was a group of cellular membrane transport protein related with water permeation. AQP5 was distributed in the respiratory epithelia from airtube to pulmonary alveolus, and involved in epithelial barrier function, membrane permeability, and water homeostasis in the respiratory epithelia.This study was designed to explore the significance of AngⅡsignal pathway and AQP5 expression in the lung of patients with congenital heart diseases and pulmonary hypertension, and to provide fresh perspective and treatment target for cure and precaution against CHD with PH.Methods:1.The study was under the guidance of the medical ethical principles. 40 cases of patients with ventricular septal defect repair were selected. The patients were divided into 4 groups according to mean pulmonary arterial pressure (mPAP) measured by pre-operative Doppler-echocardiogram: group 1 with mPAP≤25mmHg; group 2A with mPAP26～35mmHg; group 2B with mPAP36～45mmHg;group 2C with mPAP≥46mmHg). There were 10 patients in each group.2.Arterial blood of the patients in the 4 groups was sampled before the operation and AngⅡcontent in serum was measured by radio-immunity method.3.Lung tissues of the patients in each group was sampled during the operation. AT1 expression was measured by immunohistochemistry, Phosphorylated STAT1 expression was detected by Western blot, The activity of NF-κB ascertained by EMSA. AQP5 mRNA expression was inspected by RT-PCR, AQP5 protein expression was checked-out by Western blot.4.Eight patients with severe PAH were treated with oral Captopril pre-operative for two weeks,. The effects of Captopril on the Ang II signal pathway and expression of AQP5 in the lung of patients with severe PAH were investigated.Results:1.With the increase of pulmonary arterial pressure, Ang II contents in serum of all the patients tended to rise. Ang II contents in serum of Group 2A, 2B and 2C were remarkably higher than that of Group 1 (p<0.05).2.With the increase of pulmonary arterial pressure, AT1 expressions in the patients’lung tissues rose gradually. AT1 expressions of group 2A, 2B and 2C were remarkably higher than that of group 1 (p<0.05). Phosphorylated STAT1 contents in patients’lung tissues rose gradually. Phosphorylated STAT1 contents of group 2A, 2B and 2C were remarkably higher than that of group 1 (p<0.05). The activity of NF-κB in patients’lung tissues decrease gradually. The activity of NF-κB in group 2A, 2B, and 2C were remarkably lower than that of group 1 (p<0.05). AQP5 mRNA and protein expression in patients’lung tissues decline gradually. AQP5 mRNA and protein expression in group 2A, 2B and 2C were remarkably lower than that of group 1 (p<0.05).3.Captopril could inhibit the activity of Ang II signal pathway and promote the expression of AQP5 mRNA and protein in the lung tissue of patients with pulmonary arterial hypertension. The difference was significant between the control group and treatment group (p<0.05). Conclusion:1.Ang II signal pathway of patients with congenital heart disease changed remarkably with the increase of pulmonary arterial pressure. The serum Ang II level elevated. The AT1 expressions and phosphorylated STAT1 contents increased. But the activity of NF-κB decreased. The results implicated that the increasing activity of Ang II signal pathway play an important role in the pathogenesis in the genesis and development of secondary pulmonary arterial hypertension.2.With the increase of pulmonary arterial pressure, AQP5 mRNA expression and AQP5 protein content gradually decreased in the lung tissues of the patients with congenital heart disease. The results implicated that there would be some disfunction of water balance ability of alveolar epithelium in lung tissues of the patients with pulmonary hypertension. It was probably the important mechanism for congenital heart disease with pulmonary hypertension tends to pneumonedema post operation.3.In the patients with severe pulmonary hypertension, oral captopril pre-operative inhibited the activity of Ang II signal pathway of Ang II levels, AT1 expressions, phosphorylative STAT1 contents. At the same time , the NF-κB activity increased, and AQP5 mRNA and AQP5 protein expression up-regulated in the lung tissues of the patients. The results recommended that the therapy targeted on Ang II signal pathway and AQP5 expression would improve the pulmonary vascular remodeling and water homeostasis in the respiratory epithelia.更多还原