香烟烟雾促进肺动脉压力升高的作用机制的研究
发布时间：2019-05-21 11:35
【摘要】：【研究背景】 肺动脉高压(pulmonary arterial hypertension,PAH)是一种能引起右心室肥厚、右心室衰竭，并最终导致死亡的前毛细血管肺动脉床疾病。该疾病主要特征包括1)持续性肺血管收缩，2）肺动脉中层肥厚和由平滑肌细胞增生和肥厚导致的远端血管肌肉化，3）闭塞性内膜损伤。肺动脉高压是慢性阻塞性肺疾病(COPD)的重要合并症，是影响COPD患者病程的独立危险因素。轻度至中度肺动脉高压(PAH)的发病率是非常普遍的，，在终末期的慢性阻塞性肺疾病(COPD)中可达到50%。慢性阻塞性肺疾病导致的PAH的一系列改变始于疾病早期阶段内皮功能损害，其内皮功能损害与内皮衍生物如血管舒张物质（一氧化氮、前列环素）、血管收缩物质（内皮素-1）的释放受损和血管收缩-舒张失衡等密切有关。慢性阻塞性肺疾病导致的PAH是由肺动脉血管收缩和重塑引起，其肺动脉结构的改变主要是以分化差的平滑肌细胞内膜增生和弹性纤维、胶原纤维的沉积为特征。研究已证实缺氧、炎症和具有毒性作用的烟雾，是导致PAH的危险因素，这几个因素可单独或联合作用导致PAH。其中，吸烟不仅可引起慢性支气管炎、慢性阻塞性肺疾病，而且是导致PAH和慢性肺源性心脏病的原因之一。以往对COPD合并肺动脉高压发病机制的研究主要集中于缺氧导致的肺血管收缩和重建。最近的临床研究和动物实验资料显示烟雾可直接作用于肺血管系统，最终导致血管重塑和一系列血管生理反应。然而，吸烟导致肺动脉高压的具体机制尚不清楚。 细胞内游离钙离子浓度的升高，在血管平滑肌的收缩、迁移和增生过程中起着重要的作用。我们课题组和其他课题组研究证实，TRPC基因编码的蛋白是组成钙池操纵性钙通道（SOCC）和受体操纵性钙通道（ROCC）的蛋白亚单位，细胞内钙离子可以通过钙池操纵性钙内流(SOCE)来促进PASMCs的增殖，从而导致肺血管壁肥厚以及肺血管张力增加。TRPC是位于细胞膜上的一类重要的阳离子通道超家族，其在肺动脉平滑肌细胞收缩中发挥着重要的作用。研究表明，在血管平滑肌上，钾离子通道主要有电压依赖型钾离子通道（Kv）、钙激活型钾离子通道(KCa)、内向整流型钾离子通道(KATP)及双孔型钾离子通道四种通道。在这些钾离子通道中，Kv通道在调节血管平滑肌细胞膜电位、细胞内钙离子浓度及血管紧张性起着最重要作用。钾离子通道表达水平和（或）功能的降低，可令细胞膜去极化从而导致持续性的细胞内钙离子浓度的升高。而细胞钙离子浓度的升高，主要是通过以下三种途径：1）通过激活L-VDCC，2）通过促进IP3的产生增加从而刺激了肌浆网（SR）内的钙离子进入细胞浆内，（3）通过Na~+/Ca~(2+)交换从而促进钙离子内流，最终引起持续性血管收缩，这亦是构成PAH的发病机理之一。 【研究目的】 在本实验中，通过建立香烟烟雾暴露的大鼠模型及原代培养大鼠远端肺动脉平滑肌细胞，观察TRPC通道和Kv通道在香烟烟雾暴露的大鼠肺动脉平滑肌中的表达的改变；另外，通过尼古丁刺激，研究大鼠肺动脉平滑肌细胞Kv1.5和Kv2.1的mRNA表达的改变；从而探讨香烟烟雾促进大鼠肺动脉压力升高的可能机制，并了解吸烟引起肺动脉高压的机制。 【研究方法】 香烟烟雾暴露大鼠模型的建立：（1）通过直接右心测压法分别检测烟雾暴露1个月、3个月及6个月大鼠平均压(mPAP)、右心室收缩压(RVSP)、右心室肥厚指数[RV/(LV+S)]的改变及HE染色法检测肺组织病理变化，观察香烟烟雾暴露对大鼠血流动力学和肺血管结构重塑的影响；（2）应用荧光定量PCR法和免疫印迹法分别检测香烟烟雾暴露对大鼠肺动脉组织TRPC1和TRPC6的mRNA和蛋白表达的变化；另外，应用荧光定量PCR法检测香烟烟雾暴露对大鼠肺动脉组织Kv1.5和Kv2.1的mRNA表达的变化。 原代肺动脉平滑肌细胞模型的建立：（1）采用胶原酶消化法分离、培养大鼠肺动脉平滑肌细胞（PASMCs），利用InCyte细胞内钙浓度检测系统，检测香烟烟雾暴露大鼠的PASMCs的基础[Ca~(2+)]_i和SOCE；（2）应用实时荧光定量PCR法检测尼古丁（Nicotine）对大鼠远端PASMCs Kv1.5和Kv2.1的mRNA表达的影响。 【研究结果】 一、成功建立香烟烟雾暴露促进大鼠肺动脉压力升高的模型： 1、香烟烟雾暴露6个月后大鼠的mPAP、RVSP及RV/(LV+S)均有所升高，HE染色发现香烟烟雾暴露3个月后大鼠肺动脉血管壁开始出现增厚现象；随着烟雾暴露时间延长，肺动脉血管壁增厚得愈明显； 2、香烟烟雾暴露1、3和6个月均可明显上调大鼠肺动脉平滑肌TRPC1和TRPC6的mRNA和蛋白表达； 3、香烟烟雾暴露1、3和6个月均可明显下调大鼠肺动脉平滑肌Kv1.5和Kv2.1的mRNA的表达。 二、成功建立原代肺动脉平滑肌细胞模型： 1、香烟烟雾暴露3和6个月均可增加大鼠肺动脉平滑肌细胞基础钙离子浓度和钙池操纵性钙内流（SOCE）；香烟烟雾暴露6个月升高趋势较3个月明显； 2、尼古丁刺激抑制大鼠PASMCs中Kv1.5和Kv2.1的mRNA表达。 【结论】 1、香烟烟雾暴露能够促进大鼠肺动脉压力升高； 2、香烟烟雾暴露1、3和6个月均可明显上调大鼠肺动脉平滑肌TRPC1和TRPC6的mRNA和蛋白表达，同时明显下调大鼠肺动脉平滑肌Kv1.5和Kv2.1的mRNA表达，并增加SOCC介导的Ca~(2+)内流，从而增加细胞内钙离子浓度。 3、尼古丁刺激能抑制大鼠PASMCs中Kv1.5和Kv2.1的mRNA表达。
[Abstract]:[Study Background] Pulmonary arterial hypertension (PAH) is a pre-capillary pulmonary arterial bed disease that can cause right ventricular hypertrophy, right ventricular failure, and eventually lead to death. The main features of this disease include 1) persistent pulmonary vasoconstriction,2) middle-layer hypertrophy of the pulmonary artery, and distal vasospasm resulting from proliferation and hypertrophy of smooth muscle cells,3) occlusive intimal injury. Pulmonary hypertension is an important complication of chronic obstructive pulmonary disease (COPD), which is an independent risk factor that affects the course of COPD patients. A series of changes in PAH are very common in patients with chronic obstructive pulmonary disease (COPD) and are closely related to the release of endothelial derivatives such as vasodilators (nitric oxide, prostacyclin), vasoconstrictive substances (endothelin-1), and vasoconstriction-diastolic imbalance. Chronic obstructive pulmonary disease results in the contraction and remodeling of the pulmonary artery The increase of intracellular free calcium ion concentration plays an important role in the process of contraction, migration and proliferation of vascular smooth muscle. Our research group and other research group have confirmed that the protein encoded by TRPC gene is a protein subunit of the calcium pool-controlled calcium channel (SOCC) and the receptor-controlled calcium channel (ROCC). The intracellular calcium ion can promote the proliferation of PASMCs through the calcium pool-controlled calcium influx (SOCE), which can lead to the hypertrophy of the pulmonary vascular wall and the increase of the pulmonary vascular tension. The TRPC is a kind of important cation channel superfamily located on the cell membrane, which plays an important role in the contraction of the pulmonary artery smooth muscle cells. The research shows that the potassium ion channel is mainly provided with the voltage-dependent potassium ion channel (Kv), the calcium-activated potassium ion channel (KCa), the inward-rectifying type potassium ion channel (KATP) and the two-hole type potassium ion channel in these potassium ion channels. In these potassium ion channels, the Kv channel is used to regulate the membrane potential of the vascular smooth muscle cell membrane, the intracellular calcium ion concentration and the vascular tone. One of the reasons. [Objective] To observe the changes of the expression of the TRPC channel and the Kv channel in the pulmonary artery smooth muscle of rats exposed to the cigarette smoke by establishing the rat model of cigarette smoke exposure and the primary cultured rat's distal pulmonary artery smooth muscle cells. In addition, the changes of the mRNA expression of Kv1.5 and Kv2.1 in the rat pulmonary artery smooth muscle cells were studied by the nicotine stimulation. arterial high-pressure machine Methods: (1) The changes of the mean pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RV/ (LV + S)] and the change of the lung tissue were detected by the direct and right-heart pressure method. (1) The changes of the mean pressure (mPAP), the right ventricular systolic pressure (RVSP), the right ventricular hypertrophy index[RV/ (LV + S)] and the change of the pathological changes of the lung tissue were detected by the direct and right-heart pressure method, and the effects of cigarette smoke exposure on the blood flow dynamics and the remodeling of the pulmonary vascular structure were observed. (2) The changes of the expression of the mRNA and the protein of the TRPC1 and TRPC6 in the rat pulmonary artery were detected by the fluorescence quantitative PCR method and the immunoblotting method, and the exposure of the cigarette smoke to the rat pulmonary artery tissue Kv1.5 and Kv2 was detected by the fluorescence quantitative PCR method. 1. Establishment of primary pulmonary artery smooth muscle cell model: (1) Using collagenase digestion to separate and culture the rat pulmonary artery smooth muscle cells (PASMCs), using the intracellular calcium concentration detection system of InCyte cells to detect the basic[Ca ~ (2 +)] _ i and SOCE of PASMCs of cigarette smoke exposed rats; and (2) to use the real-time fluorescence quantitative PCR to detect the rat's far-end PASMCs Kv1.5 and Kv. 2.1 mR The effect of NA expression.[Results] One, successfully established cigarette smoke MPAP, RVSP and RV/ (LV + S) increased in rats after exposure to smoke for 6 months. The time of exposure was prolonged and the wall thickening of pulmonary artery was more and more obvious;2. The exposure of cigarette smoke to 1,3 and 6 months could increase the pulmonary artery smooth muscle of the rat. mRNA and protein expression of TRPC1 and TRPC6;3, cigarette smoke exposure 1,3 and 6 months could significantly reduce the rat's lung activity mRN of vein smooth muscle Kv1.5 and Kv2.1 A. Successful establishment of primary pulmonary artery smooth muscle cell model:1. The exposure of cigarette smoke 3 and 6 months can increase the calcium ion concentration in the rat pulmonary arterial smooth muscle cells and the calcium pool steerability calcium. flow (SOCE); cigarette smoke exposure for 6 months; increased trend for 3 months; nicotine stimulation Cs涓璌 mRN of Kv1.5 and Kv2.1 in PASMCs [Conclusion] 1. The exposure of cigarette smoke can promote the increase of pulmonary artery pressure in rats.2. The exposure of cigarette smoke 1,3 and 6 months can increase the mRNA and protein expression of the pulmonary artery smooth muscle TRPC1 and TRPC6 in the rat, while the mRNA and protein of the rat pulmonary artery smooth muscle Kv1.5 and Kv2.1 are significantly reduced. The Ca ~ (2 +) influx mediated by SOCC was increased, and the intracellular calcium ion concentration was increased.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2482072
[Abstract]:[Study Background] Pulmonary arterial hypertension (PAH) is a pre-capillary pulmonary arterial bed disease that can cause right ventricular hypertrophy, right ventricular failure, and eventually lead to death. The main features of this disease include 1) persistent pulmonary vasoconstriction,2) middle-layer hypertrophy of the pulmonary artery, and distal vasospasm resulting from proliferation and hypertrophy of smooth muscle cells,3) occlusive intimal injury. Pulmonary hypertension is an important complication of chronic obstructive pulmonary disease (COPD), which is an independent risk factor that affects the course of COPD patients. A series of changes in PAH are very common in patients with chronic obstructive pulmonary disease (COPD) and are closely related to the release of endothelial derivatives such as vasodilators (nitric oxide, prostacyclin), vasoconstrictive substances (endothelin-1), and vasoconstriction-diastolic imbalance. Chronic obstructive pulmonary disease results in the contraction and remodeling of the pulmonary artery The increase of intracellular free calcium ion concentration plays an important role in the process of contraction, migration and proliferation of vascular smooth muscle. Our research group and other research group have confirmed that the protein encoded by TRPC gene is a protein subunit of the calcium pool-controlled calcium channel (SOCC) and the receptor-controlled calcium channel (ROCC). The intracellular calcium ion can promote the proliferation of PASMCs through the calcium pool-controlled calcium influx (SOCE), which can lead to the hypertrophy of the pulmonary vascular wall and the increase of the pulmonary vascular tension. The TRPC is a kind of important cation channel superfamily located on the cell membrane, which plays an important role in the contraction of the pulmonary artery smooth muscle cells. The research shows that the potassium ion channel is mainly provided with the voltage-dependent potassium ion channel (Kv), the calcium-activated potassium ion channel (KCa), the inward-rectifying type potassium ion channel (KATP) and the two-hole type potassium ion channel in these potassium ion channels. In these potassium ion channels, the Kv channel is used to regulate the membrane potential of the vascular smooth muscle cell membrane, the intracellular calcium ion concentration and the vascular tone. One of the reasons. [Objective] To observe the changes of the expression of the TRPC channel and the Kv channel in the pulmonary artery smooth muscle of rats exposed to the cigarette smoke by establishing the rat model of cigarette smoke exposure and the primary cultured rat's distal pulmonary artery smooth muscle cells. In addition, the changes of the mRNA expression of Kv1.5 and Kv2.1 in the rat pulmonary artery smooth muscle cells were studied by the nicotine stimulation. arterial high-pressure machine Methods: (1) The changes of the mean pressure (mPAP), right ventricular systolic pressure (RVSP), right ventricular hypertrophy index (RV/ (LV + S)] and the change of the lung tissue were detected by the direct and right-heart pressure method. (1) The changes of the mean pressure (mPAP), the right ventricular systolic pressure (RVSP), the right ventricular hypertrophy index[RV/ (LV + S)] and the change of the pathological changes of the lung tissue were detected by the direct and right-heart pressure method, and the effects of cigarette smoke exposure on the blood flow dynamics and the remodeling of the pulmonary vascular structure were observed. (2) The changes of the expression of the mRNA and the protein of the TRPC1 and TRPC6 in the rat pulmonary artery were detected by the fluorescence quantitative PCR method and the immunoblotting method, and the exposure of the cigarette smoke to the rat pulmonary artery tissue Kv1.5 and Kv2 was detected by the fluorescence quantitative PCR method. 1. Establishment of primary pulmonary artery smooth muscle cell model: (1) Using collagenase digestion to separate and culture the rat pulmonary artery smooth muscle cells (PASMCs), using the intracellular calcium concentration detection system of InCyte cells to detect the basic[Ca ~ (2 +)] _ i and SOCE of PASMCs of cigarette smoke exposed rats; and (2) to use the real-time fluorescence quantitative PCR to detect the rat's far-end PASMCs Kv1.5 and Kv. 2.1 mR The effect of NA expression.[Results] One, successfully established cigarette smoke MPAP, RVSP and RV/ (LV + S) increased in rats after exposure to smoke for 6 months. The time of exposure was prolonged and the wall thickening of pulmonary artery was more and more obvious;2. The exposure of cigarette smoke to 1,3 and 6 months could increase the pulmonary artery smooth muscle of the rat. mRNA and protein expression of TRPC1 and TRPC6;3, cigarette smoke exposure 1,3 and 6 months could significantly reduce the rat's lung activity mRN of vein smooth muscle Kv1.5 and Kv2.1 A. Successful establishment of primary pulmonary artery smooth muscle cell model:1. The exposure of cigarette smoke 3 and 6 months can increase the calcium ion concentration in the rat pulmonary arterial smooth muscle cells and the calcium pool steerability calcium. flow (SOCE); cigarette smoke exposure for 6 months; increased trend for 3 months; nicotine stimulation Cs涓璌 mRN of Kv1.5 and Kv2.1 in PASMCs [Conclusion] 1. The exposure of cigarette smoke can promote the increase of pulmonary artery pressure in rats.2. The exposure of cigarette smoke 1,3 and 6 months can increase the mRNA and protein expression of the pulmonary artery smooth muscle TRPC1 and TRPC6 in the rat, while the mRNA and protein of the rat pulmonary artery smooth muscle Kv1.5 and Kv2.1 are significantly reduced. The Ca ~ (2 +) influx mediated by SOCC was increased, and the intracellular calcium ion concentration was increased.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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相关期刊论文 前3条
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