本文選題：低氧 + 肺動(dòng)脈高壓��； 參考：《第四軍醫(yī)大學(xué)》2007年碩士論文

【摘要】： 研究背景及目的: 慢性低氧性肺動(dòng)脈高壓(hypoxic pulmonary hypertension,HPH)是臨床常見的病理生理過(guò)程,也是慢性阻塞性肺疾病、慢性肺源性心臟病等多種心肺疾病發(fā)生發(fā)展的重要病理環(huán)節(jié)。大量的研究表明,低氧引起血管內(nèi)皮細(xì)胞一些活性物質(zhì)釋放的改變,參與了肺血管收縮及肺血管重建,從而導(dǎo)致HPH發(fā)生。慢性低氧導(dǎo)致肺動(dòng)脈高壓(pulmonary artery hypertension,PAH),卻很少引起體循環(huán)高壓即高血壓,目前傾向認(rèn)為這是肺動(dòng)脈和主動(dòng)脈對(duì)低氧的反應(yīng)性不同,對(duì)其機(jī)制鮮有報(bào)道。 一氧化氮( nitric oxide,NO )是一種內(nèi)皮源性舒張因子(endothelium-derived relax factor,EDRF),在內(nèi)皮細(xì)胞由一氧化氮合酶(nitric oxide synthase,NOS)催化L-精氨酸(L-arginine,L-Arg)產(chǎn)生。NOS存在于體內(nèi)多種細(xì)胞中,已知的亞型有三種,分別為內(nèi)皮型一氧化氮合酶(endothelial NOS,eNOS),神經(jīng)元型一氧化氮合酶(neuronal NOS,nNOS)及誘導(dǎo)型一氧化氮合酶(inducible NOS,iNOS),前兩者的產(chǎn)生受Ca2+/鈣調(diào)蛋白的調(diào)節(jié),統(tǒng)稱為結(jié)構(gòu)型一氧化氮合酶(constitutive NOS,cNOS)。已有的研究表明低氧時(shí)NOS表達(dá)量的下降使得體內(nèi)NO減少可能是發(fā)生HPH的一個(gè)機(jī)制。 肺動(dòng)脈平滑肌細(xì)胞(pulmonary arterial smooth muscle cells,PASMCs)上存在多種鉀通道(potassium channels),主要包括電壓門控性鉀通道(voltage-gated potassium channels,Kv)、Ca2+激活性鉀通道(Ca2+-activated potassium channels,KCa )及ATP敏感性鉀通道( ATP-sensitive potassium channels,KATP)等。低氧可通過(guò)抑制PASMCs的某些鉀通道活性致PASMCs膜去極化而激活電壓門控性鈣通道,從而引發(fā)細(xì)胞外Ca2+內(nèi)流,致肺動(dòng)脈平滑肌收縮,啟動(dòng)低氧性肺血管收縮。大量研究提示Kv是PASMCs上存在的各類鉀通道中與HPH發(fā)生最密切相關(guān)的一類鉀通道。Kv參與維持PASMCs的靜息膜電位(resting membrane potential, Em),低氧通過(guò)抑制Kv而減弱外向鉀電流,致PASMCs膜去極化。 本實(shí)驗(yàn)通過(guò)觀察HPH大鼠肺循環(huán)和體循環(huán)中NO含量及NOS活性是否存在差異,來(lái)說(shuō)明NO及NOS在肺動(dòng)脈和主動(dòng)脈對(duì)低氧反應(yīng)差異性中的作用。觀察硝普鈉(sodium nitroprusside,SNP,NO的外源性供體),在低氧時(shí)對(duì)體外培養(yǎng)血管平滑肌細(xì)胞Kv2.1表達(dá)的影響。 一、NO及NOS在低氧大鼠肺組織和主動(dòng)脈中的差異性及其意義 1.實(shí)驗(yàn)方法 將24只雄性SD大鼠隨機(jī)分成4組即常氧2周組、常氧3周組、低氧2周組、低氧3周組。采用間斷負(fù)壓低氧法制備大鼠低氧性肺動(dòng)脈高壓模型;右心室導(dǎo)管法測(cè)定肺動(dòng)脈收縮壓(pulmonary artery pressure,PAP);左頸總動(dòng)脈插管測(cè)量左頸總動(dòng)脈壓代表動(dòng)脈血壓(blood pressure,BP);計(jì)算右心室肥厚指數(shù)[RV/(LV+S)];采用硝酸還原酶法測(cè)定各組大鼠出、入肺血及肺組織和主動(dòng)脈勻漿中的NO含量;用化學(xué)比色法測(cè)定各組大鼠肺組織和主動(dòng)脈勻漿中NOS的活性;應(yīng)用免疫組化染色法和Western blot法觀察各組大鼠肺組織及主動(dòng)脈eNOS在蛋白質(zhì)水平表達(dá)的變化。 2.實(shí)驗(yàn)結(jié)果 低氧2周和3周組大鼠的PAP(43.4±4.4 mmHg,51.8±4.2 mmHg), [RV/(LV+S)]% ( 32.3±1.0,37.0±1.6 )均高于相應(yīng)正常對(duì)照組( 20.8±2.4 mmHg,21.8±3.9 mmHg,21.3±1.0,20.3±1.2,P0.01),且隨缺氧時(shí)間延長(zhǎng)而增高(P0.01),而BP與常氧對(duì)照組比無(wú)差別。低氧組大鼠的出、入肺血中的NO含量較其常氧對(duì)照組明顯降低(P0.01);肺組織勻漿中的NO含量、NOS活性及肺組織eNOS的表達(dá)量也較其常氧對(duì)照組明顯降低(P0.01),但這種變化在主動(dòng)脈中并不明顯;各低氧組內(nèi)主動(dòng)脈勻漿中NO含量和NOS的活性及主動(dòng)脈eNOS的表達(dá)量均高于肺組織(P0.01)。 3.小結(jié) 低氧時(shí),大鼠肺組織中NO的含量及NOS的活性均較常氧時(shí)降低,而主動(dòng)脈中二者的表達(dá)在低氧和常氧時(shí)卻沒(méi)有差別,這種差異性可能是低氧時(shí)引起肺動(dòng)脈高壓卻很少導(dǎo)致高血壓的機(jī)制之一。 二、低氧時(shí)外源性NO對(duì)PASMCs Kv2.1表達(dá)的影響 1.實(shí)驗(yàn)方法 培養(yǎng)大鼠PASMCs,實(shí)驗(yàn)分為常/低氧組,常/低氧+SNP組,常/低氧+SNP+鳥苷酸環(huán)化酶抑制劑組;MTT比色測(cè)定SNP對(duì)PASMCs增殖的影響;Western blot法檢測(cè)Kv2.1的表達(dá);RT-PCR法檢測(cè)Kv2.1mRNA水平表達(dá)的變化。 2.實(shí)驗(yàn)結(jié)果 低氧24h組PASMCs的OD值明顯高于常氧24h組,提示低氧可促進(jìn)PASMCs的增殖(P0.01);加不同濃度SNP組的OD值均低于相應(yīng)常/低氧24h組,(P0.05或P 0.01),說(shuō)明SNP在常氧和低氧時(shí)均有抑制PASMCs增殖的作用。 低氧24 h組Kv2.1在蛋白水平和mRNA水平表達(dá)較常氧24h組明顯低;加入SNP后,Kv2.1的表達(dá)增加,且這種變化在常氧和低氧組都可以看到,說(shuō)明SNP可以增加Kv2.1的表達(dá);而在加入了阻斷劑MB后,Kv2.1的表達(dá)又有所下降,表明SNP對(duì)Kv2.1的影響是通過(guò)cGMP途徑實(shí)現(xiàn)的。 3.小結(jié) SNP能有效的抑制PASMCs的增殖,緩解低氧性肺血管收縮,而這種作用可能是通過(guò)其對(duì)鉀通道的影響實(shí)現(xiàn)的,這為SNP治療HPH進(jìn)一步提供理論依據(jù)。 結(jié)論 低氧時(shí)肺循環(huán)NO系統(tǒng)受抑制是體肺循環(huán)對(duì)低氧反應(yīng)性差異的原因之一,也是HPH發(fā)生發(fā)展的機(jī)制之一;本研究證實(shí)外源性NO通過(guò)cGMP途徑,增加Kv2.1的表達(dá),這可能與其抑制低氧性PASMCs增殖和肺動(dòng)脈收縮有關(guān)。
[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.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363

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本文編號(hào)：2053427