低氧时NO及NOS在体肺循环中表达的差异性及外源性NO对Kv2.1的影响

发布时间：2018-06-22 16:27

本文选题：低氧 + 肺动脉高压　；参考：《第四军医大学》2007年硕士论文

【摘要】： 研究背景及目的: 慢性低氧性肺动脉高压(hypoxic pulmonary hypertension,HPH)是临床常见的病理生理过程,也是慢性阻塞性肺疾病、慢性肺源性心脏病等多种心肺疾病发生发展的重要病理环节。大量的研究表明,低氧引起血管内皮细胞一些活性物质释放的改变,参与了肺血管收缩及肺血管重建,从而导致HPH发生。慢性低氧导致肺动脉高压(pulmonary artery hypertension,PAH),却很少引起体循环高压即高血压,目前倾向认为这是肺动脉和主动脉对低氧的反应性不同,对其机制鲜有报道。一氧化氮( nitric oxide,NO )是一种内皮源性舒张因子(endothelium-derived relax factor,EDRF),在内皮细胞由一氧化氮合酶(nitric oxide synthase,NOS)催化L-精氨酸(L-arginine,L-Arg)产生。NOS存在于体内多种细胞中,已知的亚型有三种,分别为内皮型一氧化氮合酶(endothelial NOS,eNOS),神经元型一氧化氮合酶(neuronal NOS,nNOS)及诱导型一氧化氮合酶(inducible NOS,iNOS),前两者的产生受Ca2+/钙调蛋白的调节,统称为结构型一氧化氮合酶(constitutive NOS,cNOS)。已有的研究表明低氧时NOS表达量的下降使得体内NO减少可能是发生HPH的一个机制。肺动脉平滑肌细胞(pulmonary arterial smooth muscle cells,PASMCs)上存在多种钾通道(potassium channels),主要包括电压门控性钾通道(voltage-gated potassium channels,Kv)、Ca2+激活性钾通道(Ca2+-activated potassium channels,KCa )及ATP敏感性钾通道( ATP-sensitive potassium channels,KATP)等。低氧可通过抑制PASMCs的某些钾通道活性致PASMCs膜去极化而激活电压门控性钙通道,从而引发细胞外Ca2+内流,致肺动脉平滑肌收缩,启动低氧性肺血管收缩。大量研究提示Kv是PASMCs上存在的各类钾通道中与HPH发生最密切相关的一类钾通道。Kv参与维持PASMCs的静息膜电位(resting membrane potential, Em),低氧通过抑制Kv而减弱外向钾电流,致PASMCs膜去极化。本实验通过观察HPH大鼠肺循环和体循环中NO含量及NOS活性是否存在差异,来说明NO及NOS在肺动脉和主动脉对低氧反应差异性中的作用。观察硝普钠(sodium nitroprusside,SNP,NO的外源性供体),在低氧时对体外培养血管平滑肌细胞Kv2.1表达的影响。一、NO及NOS在低氧大鼠肺组织和主动脉中的差异性及其意义 1.实验方法将24只雄性SD大鼠随机分成4组即常氧2周组、常氧3周组、低氧2周组、低氧3周组。采用间断负压低氧法制备大鼠低氧性肺动脉高压模型;右心室导管法测定肺动脉收缩压(pulmonary artery pressure,PAP);左颈总动脉插管测量左颈总动脉压代表动脉血压(blood pressure,BP);计算右心室肥厚指数[RV/(LV+S)];采用硝酸还原酶法测定各组大鼠出、入肺血及肺组织和主动脉匀浆中的NO含量;用化学比色法测定各组大鼠肺组织和主动脉匀浆中NOS的活性;应用免疫组化染色法和Western blot法观察各组大鼠肺组织及主动脉eNOS在蛋白质水平表达的变化。 2.实验结果低氧2周和3周组大鼠的PAP(43.4±4.4 mmHg,51.8±4.2 mmHg), [RV/(LV+S)]% ( 32.3±1.0,37.0±1.6 )均高于相应正常对照组( 20.8±2.4 mmHg,21.8±3.9 mmHg,21.3±1.0,20.3±1.2,P0.01),且随缺氧时间延长而增高(P0.01),而BP与常氧对照组比无差别。低氧组大鼠的出、入肺血中的NO含量较其常氧对照组明显降低(P0.01);肺组织匀浆中的NO含量、NOS活性及肺组织eNOS的表达量也较其常氧对照组明显降低(P0.01),但这种变化在主动脉中并不明显;各低氧组内主动脉匀浆中NO含量和NOS的活性及主动脉eNOS的表达量均高于肺组织(P0.01)。 3.小结低氧时,大鼠肺组织中NO的含量及NOS的活性均较常氧时降低,而主动脉中二者的表达在低氧和常氧时却没有差别,这种差异性可能是低氧时引起肺动脉高压却很少导致高血压的机制之一。二、低氧时外源性NO对PASMCs Kv2.1表达的影响 1.实验方法培养大鼠PASMCs,实验分为常/低氧组,常/低氧+SNP组,常/低氧+SNP+鸟苷酸环化酶抑制剂组;MTT比色测定SNP对PASMCs增殖的影响;Western blot法检测Kv2.1的表达;RT-PCR法检测Kv2.1mRNA水平表达的变化。 2.实验结果低氧24h组PASMCs的OD值明显高于常氧24h组,提示低氧可促进PASMCs的增殖(P0.01);加不同浓度SNP组的OD值均低于相应常/低氧24h组,(P0.05或P 0.01),说明SNP在常氧和低氧时均有抑制PASMCs增殖的作用。低氧24 h组Kv2.1在蛋白水平和mRNA水平表达较常氧24h组明显低;加入SNP后,Kv2.1的表达增加,且这种变化在常氧和低氧组都可以看到,说明SNP可以增加Kv2.1的表达;而在加入了阻断剂MB后,Kv2.1的表达又有所下降,表明SNP对Kv2.1的影响是通过cGMP途径实现的。 3.小结 SNP能有效的抑制PASMCs的增殖,缓解低氧性肺血管收缩,而这种作用可能是通过其对钾通道的影响实现的,这为SNP治疗HPH进一步提供理论依据。结论低氧时肺循环NO系统受抑制是体肺循环对低氧反应性差异的原因之一,也是HPH发生发展的机制之一;本研究证实外源性NO通过cGMP途径,增加Kv2.1的表达,这可能与其抑制低氧性PASMCs增殖和肺动脉收缩有关。
[Abstract]:Research background and purpose:
Chronic hypoxic pulmonary hypertension (hypoxic pulmonary hypertension, HPH) is a common pathophysiological process. It is also an important pathological link in the development of chronic obstructive pulmonary disease, chronic pulmonary heart disease and many other cardiopulmonary diseases. A large number of studies have shown that hypoxia induces the release of some active substances in vascular endothelial cells. It participates in pulmonary vasoconstriction and pulmonary vascular reconstruction, which leads to the occurrence of HPH. Chronic hypoxia leads to pulmonary hypertension (pulmonary artery hypertension, PAH), but it rarely causes systemic circulation high pressure, which is currently considered to be a different reaction of pulmonary artery and aorta to hypoxia, and the mechanism is rarely reported.
Nitric oxide (NO) is an endothelium-derived diastolic factor (endothelium-derived relax factor, EDRF). In endothelial cells, the nitric oxide synthase (nitric oxide synthase, NOS) catalyzes the existence of L- arginine in a variety of cells. There are three known subtypes, endothelial nitric oxide, respectively. Endothelial NOS (eNOS), neuronal nitric oxide synthase (neuronal NOS, nNOS) and inducible nitric oxide synthase (inducible NOS, iNOS). The first two are regulated by Ca2+/ calmodulin, collectively known as the structural nitric oxide synthase (constitutive NOS,). Internal NO reduction may be a mechanism for the occurrence of HPH.
There are a variety of potassium channels (potassium channels) on pulmonary arterial smooth muscle cells (PASMCs), which mainly include voltage gated potassium channels (voltage-gated potassium channels, Kv), activated potassium channels and sensitive potassium channels. Um channels, KATP) and so on. Hypoxia can activate the voltage gated calcium channel by inhibiting the activity of some potassium channels of the PASMCs and activating the voltage gated calcium channel, thus triggering the extracellular Ca2+ flow, causing the contraction of the pulmonary artery smooth muscle and starting the hypoxic pulmonary vasoconstriction. A large number of studies suggest that Kv is the most closely associated with HPH in all kinds of potassium channels existing on PASMCs. A related type of potassium channel.Kv participates in maintaining the resting membrane potential of PASMCs (resting membrane potential, Em). Hypoxia reduces the outward potassium current by inhibiting the Kv and causes depolarization of PASMCs membrane.
By observing the difference of NO content and NOS activity in the pulmonary circulation and body circulation of HPH rats, the effect of NO and NOS on the difference of hypoxic reaction between the pulmonary artery and the aorta was demonstrated. The effects of sodium nitroprusside (sodium nitroprusside, SNP, NO exogenous donor) and the expression of Kv2.1 expression of vascular smooth muscle cells in vitro were observed in hypoxia. Ringing.
The difference between NO and NOS in lung tissue and aorta of hypoxic rats and its significance
1. experimental method
24 male SD rats were randomly divided into 4 groups, namely, 2 weeks of normal oxygen, 3 weeks of normal oxygen, 2 weeks of hypoxia and 3 weeks of hypoxia. The rat model of hypoxic pulmonary hypertension was prepared by intermittent negative pressure hypoxia; right ventricular catheter method was used to measure the systolic pressure of pulmonary artery (pulmonary artery pressure, PAP); left carotid artery intubation was used to measure the left carotid artery pressure to represent the artery The blood pressure (blood pressure, BP) and the right ventricular hypertrophy index [RV/ (LV+S)] were calculated. The nitrate reductase was used to determine the NO content in the lung and lung tissue and the aortic homogenate, and the activity of NOS in the lung tissue and the aorta homogenate of each group was measured by chemical colorimetry, and the immunohistochemical staining and Western blot method were used to observe each group. Changes of eNOS expression in lung tissue and aorta of rats in protein group.
2. experimental results
PAP (43.4 + 4.4 mmHg, 51.8 + 4.2 mmHg), [RV/ (LV+S))% (32.3 + 1.0,37.0 + 1.6) of rats in 2 and 3 weeks of hypoxia were higher than that in the corresponding control group (20.8 + 2.4 mmHg, 21.8 + 3.9 mmHg, 21.3 + 1.0,20.3 +, P0.01), and increased with the prolonged hypoxia time (P0.01), but there was no difference between the BP and the normal oxygen control group. The rats in the hypoxia group were out of the lung blood. The content of NO in the control group was significantly lower than that of the normal oxygen control group (P0.01), and the NO content in the lung homogenate, the NOS activity and the expression of eNOS in the lung tissue were also significantly lower than those of the normal oxygen control group (P0.01), but this change was not obvious in the aorta, and the NO content and NOS activity in the aortic homogenate and the expression of eNOS in the aorta in the hypoxic groups were both high. In the lung tissue (P0.01).
3. summary
When hypoxia, the content of NO and the activity of NOS in the lung tissue of rats are lower than that of the normal oxygen, but the expression of the two in the aorta is not different when hypoxia and normoxia, which may be one of the mechanisms that cause pulmonary hypertension but rarely lead to hypertension.
Two, the effect of exogenous NO on the expression of PASMCs Kv2.1 in hypoxia.
1. experimental method
Rat PASMCs was cultured. The experiment was divided into normal / hypoxia group, normal / hypoxic +SNP group, constant / hypoxia +SNP+ guanosine cyclase inhibitor group; MTT colorimetric assay was used to determine the effect of SNP on PASMCs proliferation; Western blot method was used to detect the expression of Kv2.1; RT-PCR assay was used to detect the Kv2.1mRNA level expression.
2. experimental results
The OD value of PASMCs in the hypoxia 24h group was significantly higher than that of the normal oxygen 24h group, suggesting that hypoxia could promote the proliferation of PASMCs (P0.01), and the OD values of SNP groups with different concentrations were lower than those of the corresponding normal / low oxygen 24h group (P0.05 or P 0.01), indicating that SNP could inhibit PASMCs proliferation in normal oxygen and hypoxia.
The expression of Kv2.1 at protein level and mRNA level in the hypoxia 24 h group was significantly lower than that in the normal oxygen 24h group, and the expression of Kv2.1 increased after adding SNP, and this change was seen in the normoxic and hypoxia groups, indicating that SNP could increase the expression of Kv2.1, and the expression of Kv2.1 decreased after the addition of the blocker MB, indicating that SNP on Kv2.1 was passed. The way is realized.
3. summary
SNP can effectively inhibit the proliferation of PASMCs and alleviate hypoxic pulmonary vasoconstriction, which may be achieved through its effect on the potassium channel, which provides a further theoretical basis for the treatment of HPH by SNP.
conclusion
The inhibition of pulmonary circulation NO system in hypoxic pulmonary circulation is one of the reasons for the difference of hypoxic responsiveness in the lung circulation and one of the mechanisms of HPH development. This study confirms that exogenous NO increases the expression of Kv2.1 through the cGMP pathway, which may be related to the inhibition of hypoxic PASMCs proliferation and pulmonary artery contraction.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363

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