香煙煙霧促進(jìn)肺動(dòng)脈壓力升高的作用機(jī)制的研究
發(fā)布時(shí)間：2019-05-21 11:35
【摘要】：【研究背景】 肺動(dòng)脈高壓(pulmonary arterial hypertension,PAH)是一種能引起右心室肥厚、右心室衰竭，并最終導(dǎo)致死亡的前毛細(xì)血管肺動(dòng)脈床疾病。該疾病主要特征包括1)持續(xù)性肺血管收縮，2）肺動(dòng)脈中層肥厚和由平滑肌細(xì)胞增生和肥厚導(dǎo)致的遠(yuǎn)端血管肌肉化，3）閉塞性內(nèi)膜損傷。肺動(dòng)脈高壓是慢性阻塞性肺疾病(COPD)的重要合并癥，是影響COPD患者病程的獨(dú)立危險(xiǎn)因素。輕度至中度肺動(dòng)脈高壓(PAH)的發(fā)病率是非常普遍的，，在終末期的慢性阻塞性肺疾病(COPD)中可達(dá)到50%。慢性阻塞性肺疾病導(dǎo)致的PAH的一系列改變始于疾病早期階段內(nèi)皮功能損害，其內(nèi)皮功能損害與內(nèi)皮衍生物如血管舒張物質(zhì)（一氧化氮、前列環(huán)素）、血管收縮物質(zhì)（內(nèi)皮素-1）的釋放受損和血管收縮-舒張失衡等密切有關(guān)。慢性阻塞性肺疾病導(dǎo)致的PAH是由肺動(dòng)脈血管收縮和重塑引起，其肺動(dòng)脈結(jié)構(gòu)的改變主要是以分化差的平滑肌細(xì)胞內(nèi)膜增生和彈性纖維、膠原纖維的沉積為特征。研究已證實(shí)缺氧、炎癥和具有毒性作用的煙霧，是導(dǎo)致PAH的危險(xiǎn)因素，這幾個(gè)因素可單獨(dú)或聯(lián)合作用導(dǎo)致PAH。其中，吸煙不僅可引起慢性支氣管炎、慢性阻塞性肺疾病，而且是導(dǎo)致PAH和慢性肺源性心臟病的原因之一。以往對(duì)COPD合并肺動(dòng)脈高壓發(fā)病機(jī)制的研究主要集中于缺氧導(dǎo)致的肺血管收縮和重建。最近的臨床研究和動(dòng)物實(shí)驗(yàn)資料顯示煙霧可直接作用于肺血管系統(tǒng)，最終導(dǎo)致血管重塑和一系列血管生理反應(yīng)。然而，吸煙導(dǎo)致肺動(dòng)脈高壓的具體機(jī)制尚不清楚。 細(xì)胞內(nèi)游離鈣離子濃度的升高，在血管平滑肌的收縮、遷移和增生過程中起著重要的作用。我們課題組和其他課題組研究證實(shí)，TRPC基因編碼的蛋白是組成鈣池操縱性鈣通道（SOCC）和受體操縱性鈣通道（ROCC）的蛋白亞單位，細(xì)胞內(nèi)鈣離子可以通過鈣池操縱性鈣內(nèi)流(SOCE)來促進(jìn)PASMCs的增殖，從而導(dǎo)致肺血管壁肥厚以及肺血管張力增加。TRPC是位于細(xì)胞膜上的一類重要的陽離子通道超家族，其在肺動(dòng)脈平滑肌細(xì)胞收縮中發(fā)揮著重要的作用。研究表明，在血管平滑肌上，鉀離子通道主要有電壓依賴型鉀離子通道（Kv）、鈣激活型鉀離子通道(KCa)、內(nèi)向整流型鉀離子通道(KATP)及雙孔型鉀離子通道四種通道。在這些鉀離子通道中，Kv通道在調(diào)節(jié)血管平滑肌細(xì)胞膜電位、細(xì)胞內(nèi)鈣離子濃度及血管緊張性起著最重要作用。鉀離子通道表達(dá)水平和（或）功能的降低，可令細(xì)胞膜去極化從而導(dǎo)致持續(xù)性的細(xì)胞內(nèi)鈣離子濃度的升高。而細(xì)胞鈣離子濃度的升高，主要是通過以下三種途徑：1）通過激活L-VDCC，2）通過促進(jìn)IP3的產(chǎn)生增加從而刺激了肌漿網(wǎng)（SR）內(nèi)的鈣離子進(jìn)入細(xì)胞漿內(nèi)，（3）通過Na~+/Ca~(2+)交換從而促進(jìn)鈣離子內(nèi)流，最終引起持續(xù)性血管收縮，這亦是構(gòu)成PAH的發(fā)病機(jī)理之一。 【研究目的】 在本實(shí)驗(yàn)中，通過建立香煙煙霧暴露的大鼠模型及原代培養(yǎng)大鼠遠(yuǎn)端肺動(dòng)脈平滑肌細(xì)胞，觀察TRPC通道和Kv通道在香煙煙霧暴露的大鼠肺動(dòng)脈平滑肌中的表達(dá)的改變；另外，通過尼古丁刺激，研究大鼠肺動(dòng)脈平滑肌細(xì)胞Kv1.5和Kv2.1的mRNA表達(dá)的改變；從而探討香煙煙霧促進(jìn)大鼠肺動(dòng)脈壓力升高的可能機(jī)制，并了解吸煙引起肺動(dòng)脈高壓的機(jī)制。 【研究方法】 香煙煙霧暴露大鼠模型的建立：（1）通過直接右心測壓法分別檢測煙霧暴露1個(gè)月、3個(gè)月及6個(gè)月大鼠平均壓(mPAP)、右心室收縮壓(RVSP)、右心室肥厚指數(shù)[RV/(LV+S)]的改變及HE染色法檢測肺組織病理變化，觀察香煙煙霧暴露對(duì)大鼠血流動(dòng)力學(xué)和肺血管結(jié)構(gòu)重塑的影響；（2）應(yīng)用熒光定量PCR法和免疫印跡法分別檢測香煙煙霧暴露對(duì)大鼠肺動(dòng)脈組織TRPC1和TRPC6的mRNA和蛋白表達(dá)的變化；另外，應(yīng)用熒光定量PCR法檢測香煙煙霧暴露對(duì)大鼠肺動(dòng)脈組織Kv1.5和Kv2.1的mRNA表達(dá)的變化。 原代肺動(dòng)脈平滑肌細(xì)胞模型的建立：（1）采用膠原酶消化法分離、培養(yǎng)大鼠肺動(dòng)脈平滑肌細(xì)胞（PASMCs），利用InCyte細(xì)胞內(nèi)鈣濃度檢測系統(tǒng)，檢測香煙煙霧暴露大鼠的PASMCs的基礎(chǔ)[Ca~(2+)]_i和SOCE；（2）應(yīng)用實(shí)時(shí)熒光定量PCR法檢測尼古�。∟icotine）對(duì)大鼠遠(yuǎn)端PASMCs Kv1.5和Kv2.1的mRNA表達(dá)的影響。 【研究結(jié)果】 一、成功建立香煙煙霧暴露促進(jìn)大鼠肺動(dòng)脈壓力升高的模型： 1、香煙煙霧暴露6個(gè)月后大鼠的mPAP、RVSP及RV/(LV+S)均有所升高，HE染色發(fā)現(xiàn)香煙煙霧暴露3個(gè)月后大鼠肺動(dòng)脈血管壁開始出現(xiàn)增厚現(xiàn)象；隨著煙霧暴露時(shí)間延長，肺動(dòng)脈血管壁增厚得愈明顯； 2、香煙煙霧暴露1、3和6個(gè)月均可明顯上調(diào)大鼠肺動(dòng)脈平滑肌TRPC1和TRPC6的mRNA和蛋白表達(dá)； 3、香煙煙霧暴露1、3和6個(gè)月均可明顯下調(diào)大鼠肺動(dòng)脈平滑肌Kv1.5和Kv2.1的mRNA的表達(dá)。 二、成功建立原代肺動(dòng)脈平滑肌細(xì)胞模型： 1、香煙煙霧暴露3和6個(gè)月均可增加大鼠肺動(dòng)脈平滑肌細(xì)胞基礎(chǔ)鈣離子濃度和鈣池操縱性鈣內(nèi)流（SOCE）；香煙煙霧暴露6個(gè)月升高趨勢較3個(gè)月明顯； 2、尼古丁刺激抑制大鼠PASMCs中Kv1.5和Kv2.1的mRNA表達(dá)。 【結(jié)論】 1、香煙煙霧暴露能夠促進(jìn)大鼠肺動(dòng)脈壓力升高； 2、香煙煙霧暴露1、3和6個(gè)月均可明顯上調(diào)大鼠肺動(dòng)脈平滑肌TRPC1和TRPC6的mRNA和蛋白表達(dá)，同時(shí)明顯下調(diào)大鼠肺動(dòng)脈平滑肌Kv1.5和Kv2.1的mRNA表達(dá)，并增加SOCC介導(dǎo)的Ca~(2+)內(nèi)流，從而增加細(xì)胞內(nèi)鈣離子濃度。 3、尼古丁刺激能抑制大鼠PASMCs中Kv1.5和Kv2.1的mRNA表達(dá)。
[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.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363
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