發(fā)布時間：2018-06-04 12:09

  本文選題：雌激素 + 內(nèi)源性雌激素　； 參考：《第四軍醫(yī)大學(xué)》2014年博士論文

【摘要】：研究背景： 低氧性肺動脈高壓（Hypoxic pulmonary hypertension, HPH）是肺動脈高壓（Pulmonary hypertension, PH）中的一個亞型，常見于罹患慢性阻塞性肺疾病與久居高原的人群。HPH以血管壁的增厚與重建為主要特征，其病理生理學(xué)基礎(chǔ)包括低氧性肺血管收縮（Hypoxic pulmonary vasoconstriction, HPV），肺動脈平滑肌細胞的肥大增殖，以及后期的肺動脈血管的重構(gòu)（Pulmonary vascular remodeling, PVR）和肺動脈壓力升高。肺動脈血管的結(jié)構(gòu)重構(gòu)和晚期右心室功能衰竭是PH患者病情惡化及導(dǎo)致死亡的主要原因。小動脈壁的肌層增厚與無肌層小動脈肌化是HPH病理學(xué)改變的 主要特征。 研究發(fā)現(xiàn)HPH的發(fā)病存在有明顯的性別差異，高原低氧環(huán)境中女性發(fā)病率遠低于男性，且女性患者臨床表現(xiàn)也相對較輕；后來研究發(fā)現(xiàn)雌激素在其中發(fā)揮了舒張血管、降低肺動脈壓力等重要作用。因此，進一步揭示雌激素對HPH的保護作用及機制，尋找新的安全有效的外源性雌激素，將為早期干預(yù)和治療PH提供新的作用靶點及手段。本實驗擬從動物整體、器官與細胞水平觀察內(nèi)源性雌激素、外源性雌激素和植物性雌激素對HPH的作用并深入探討其作用機制。實驗?zāi)康模?從整體、血管和細胞水平三個層次探討內(nèi)源性、外源性及植物雌激素對HPH的防治作用，進一步從細胞周期、炎癥、氧化應(yīng)激等方面揭示雌激素在防治HPH作用中的機制： （1）探討內(nèi)源性雌激素在HPH發(fā)生過程中的作用與機制。 （2）研究AKT/Skp2/P27kip1途徑在雌激素防治HPH中的作用。 （3）探究植物性雌激素在防治HPH中的作用及其作用機制。 實驗方法： 一、觀察并探討內(nèi)源性雌激素在HPH中的作用及機制 我們分別在動物、肺動脈血管環(huán)和肺動脈平滑肌細胞水平進行研究。首先將成年雌性、雄性、懷孕及卵巢切除的SD大鼠（Sprague-Dawley rats）隨機分為常氧組和低氧組等不同的8個組。常氧組置于自然條件（西安地區(qū)大氣壓力約為718mmHg，pO2為150.6mmHg），低氧組置于低氧艙中減壓至380mmHg（相應(yīng)的pO2減至79.6mmHg，相當于海拔5000米高度的氧分壓），連續(xù)21天復(fù)制HPH的大鼠模型，觀察對比各組大鼠的血流動力學(xué)指標，如右心室收縮壓（Right ventricular systolic pressure,RVSP）、平均肺動脈壓（mean pulmonary arterial pressure, mPAP）及肺動脈的病理結(jié)構(gòu)變化等指標。其次，將雄性和不同動情周期的雌性大鼠麻醉后取肺動脈第3級血管（血管外徑300μm），將血管條浸置于37℃的Krebs-Henseleit液中并通有混合氣（95%O2:5%CO2）條件下60分鐘，再更換為低氧混合氣（95%N2:5%O2），分別用雌激素受體調(diào)節(jié)劑雷洛昔芬和MAPK抑制劑U0126（MAPK為雌激素膜受體GPR30的下游通路）進行干預(yù)，并用放射性免疫法測定各組大鼠的內(nèi)源性雌激素的水平，觀察內(nèi)源性雌激素的生理性變化在急性低氧條件下對肺動脈血管收縮的作用。最后，采用取自不同性別和動情周期的大鼠肺動脈，原代培養(yǎng)平滑肌細胞，并于2%低氧條件下培養(yǎng)，分別用雷洛昔芬和U0126進行干預(yù)，[3H]-胸腺嘧啶脫氧核苷摻入法測定低氧引起的肺動脈平滑肌細胞增殖，進而觀察內(nèi)源性雌激素水平與低氧肺動脈平滑肌細胞增殖的關(guān)系。 二、觀察外源性雌激素在HPH中的作用及探討P27kip1是否參與其內(nèi)在保護機制 我們在復(fù)制雄性大鼠HPH模型的基礎(chǔ)上，同時給予外源性雌激素β-雌二醇干預(yù)，觀察大鼠HPH相關(guān)的血流動力學(xué)參數(shù)與肺小動脈的病理形態(tài)學(xué)改變，通過免疫印跡實驗、逆轉(zhuǎn)錄PCR方法測定了肺組織與肺動脈平滑肌細胞中與細胞周期相關(guān)的AKT/Skp2/P27kip1等關(guān)鍵分子，并結(jié)合β-雌二醇對低氧導(dǎo)致的肺動脈平滑肌細胞增殖的影響，在整體和細胞水平探討外源性雌激素是否通過調(diào)節(jié)細胞周期而發(fā)揮對HPH的保護作用。 三、觀察并初步闡釋植物性雌激素白藜蘆醇對HPH的保護作用與機制 為了尋找防治效果好，副作用小的化合物，，我們進一步觀察植物雌激素白藜蘆醇對HPH的防治作用。在整體水平，復(fù)制雄性大鼠HPH模型，同時給予植物雌激素白藜蘆醇（40mg/kg/day，灌胃），同樣觀察大鼠HPH相關(guān)的血流動力學(xué)參數(shù)與肺小動脈的病理形態(tài)學(xué)改變，采用免疫組織化學(xué)染色方法觀察大鼠肺動脈血管組織周圍的炎癥細胞浸潤，Realtime-PCR檢測肺組織中IL-6、TNF-α等炎癥介質(zhì)的表達，并采用免疫印跡實驗等方法觀察與炎癥和氧化應(yīng)激相關(guān)的HIF-1α、NF-κB、Nrf-2/Trx-1、H2O2、GSH、SOD等各關(guān)鍵分子的表達變化；在細胞水平低氧培養(yǎng)肺動脈平滑肌細胞的同時，給予不同濃度的植物雌激素白藜蘆醇進行干預(yù)，同時給予雌激素受體抑制劑ICI182780阻斷雌激素受體，采用WST-1實驗觀察植物雌激素白藜蘆醇對低氧肺動脈平滑肌細胞增殖的影響，另外通過使用HIF-1α抑制劑KC7F2抑制HIF-1α，并分別用AKT和ERK抑制劑LY294002和PD98059抑制AKT通路與ERK通路，進一步從炎癥和氧化應(yīng)激兩方面揭示植物雌激素白藜蘆醇對大鼠HPH和低氧性肺動脈平滑肌增殖的影響及其機制。 實驗結(jié)果： 1）內(nèi)源性雌激素的生理性變化可以影響大鼠HPH的發(fā)生發(fā)展，雌激素水平高的雌性低氧組大鼠比雄性低氧組大鼠表現(xiàn)出較輕的RVSP、mPAP等血流動力學(xué)指標的升高與肺小動脈壁增厚等表現(xiàn)，其中雌激素水平最高的孕鼠HPH參數(shù)變化最輕；而卵巢切除低氧組大鼠的雌激素水平接近于雄性大鼠，其RVSP、mPAP升高幅度及肺小動脈壁增厚等表現(xiàn)也接近于雄性低氧組。離體血管實驗顯示，與雄性大鼠和動情間期大鼠相比，取自雌激素水平較高的動情前期大鼠的肺動脈血管表現(xiàn)出較輕的HPV反應(yīng)，MAPK抑制劑U0126增加了HPV反應(yīng)。細胞水平實驗進一步表明來自雌激素水平較高的動情前期大鼠的肺動脈平滑肌細胞表現(xiàn)出較輕的增殖反應(yīng)，雷洛昔芬與U0126干預(yù)在一定程度上增強了低氧導(dǎo)致的肺動脈平滑肌細胞增殖，雷洛昔芬與U0126同時干預(yù)時表現(xiàn)出疊加效應(yīng)，明顯增加了低氧導(dǎo)致的肺動脈平滑肌細胞增殖。 2）低氧導(dǎo)致大鼠RVSP和右心室重構(gòu)指數(shù)RVHI的明顯升高、肺小動脈血管壁的顯著增厚；并且伴隨了肺組織中細胞周期抑制因子P27kip1的表達明顯減少，以及P27kip1特異性降解酶S期激酶蛋白2（Skp-2）與磷酸化AKT蛋白水平表達的明顯升高。外源性雌激素β-雌二醇有效減輕低氧導(dǎo)致的大鼠RVSP和右心室重構(gòu)指數(shù)RVHI的升高，減輕了肺小動脈血管壁的增厚，在減少磷酸化AKT和Skp-2表達的同時升高細胞周期抑制因子P27kip1的表達；細胞實驗進一步驗證大體實驗結(jié)果，并顯示β-雌二醇能夠顯著降低低氧誘導(dǎo)的肺動脈平滑肌細胞的增殖。 3）植物性雌激素白藜蘆醇明顯抑制大鼠HPH的發(fā)生發(fā)展，減輕低氧導(dǎo)致的右心室壓力升高與右心室重構(gòu)，同時減少低氧引起的HIF-1α、NF-κB在肺組織中的表達增加，減輕了炎癥細胞在肺動脈血管周圍的浸潤和肺組織中H2O2的生成，減少低氧引起的IL-6、TNF-α等炎癥因子的mRNA水平的升高，增加了Nrf-2、Trx-1、GSH與SOD等抗氧化應(yīng)激因子的表達；細胞實驗表明白藜蘆醇以濃度依賴的方式減少低氧導(dǎo)致的HIF-1α在肺動脈平滑肌細胞中的表達，并且抑制HIF-1α的表達的同時可以有效抑制低氧肺動脈平滑肌細胞增殖，雌激素受體抑制劑ICI182780不能阻斷白藜蘆醇抑制低氧肺血管平滑肌增殖的作用，白藜蘆醇在抑制低氧導(dǎo)致的肺動脈平滑肌細胞增殖的同時也抑制磷酸化AKT和ERK的表達。 結(jié)論： 1）內(nèi)源性雌激素在大鼠HPH發(fā)生過程中發(fā)揮了拮抗性作用。可能通過非基因組作用途徑（GPR30受體途徑）降低HPV和基因組途徑抑制PVR和PASMCs的增殖從而發(fā)揮其拮抗HPH的作用。 2）外源性雌激素β-雌二醇能有效改善大鼠HPH，通過調(diào)節(jié)AKT/Skp2/P27kip1途徑抑制肺動脈平滑肌細胞的增殖是其發(fā)揮作用的重要機制之一。 3）植物雌激素白藜蘆醇能減輕HPH，該作用與其抑制HIF-1α途徑和降低炎癥反應(yīng)，以及激活Nrf-2/Trx-1通路，減少氧化應(yīng)激反應(yīng)有關(guān)，其作用可能為非雌激素受體依賴的方式，但與抑制AKT和ERK通路有關(guān)。
[Abstract]:Research background:
Hypoxic pulmonary hypertension (Hypoxic pulmonary hypertension, HPH) is a subtype of pulmonary hypertension (Pulmonary hypertension, PH). It is common in chronic obstructive pulmonary disease and people who live on high altitude..HPH is the main feature of vascular wall thickening and reconstruction, and its pathophysiological basis includes hypoxic pulmonary vasoconstriction (Hypox). IC pulmonary vasoconstriction, HPV), hypertrophy and proliferation of pulmonary artery smooth muscle cells, and later remodeling of pulmonary artery (Pulmonary vascular remodeling, PVR) and pulmonary artery pressure. Structural remodeling of pulmonary artery and late right ventricular failure are the main reasons for the deterioration of the patient's condition and the leading cause of death. Myometrial thickening and myocutaneous arterioles are the pathological changes of HPH.
Main features.
The study found that there was a significant gender difference in the incidence of HPH, the incidence of women in high altitude hypoxia was far lower than that of men, and the clinical manifestations of women were relatively light. Later, the study found that estrogen played an important role in diastolic blood vessels and decreased the pressure of pulmonary artery. Therefore, the protective effect of estrogen on HPH was further revealed. And the mechanism of finding new safe and effective exogenous estrogen will provide new targets and means for early intervention and treatment of PH. This experiment is to observe the role of endogenous estrogen, exogenous estrogen and phytoestrogen to HPH and explore the mechanism of its action from the animal whole, organ and cell level.
The effects of endogenous, exogenous and phytoestrogens on the prevention and control of HPH were discussed from three levels, including the whole, blood vessel and cell level, and the mechanisms of estrogen in the prevention and control of HPH were revealed from cell cycle, inflammation and oxidative stress.
(1) to explore the role and mechanism of endogenous estrogen in the pathogenesis of HPH.
(2) to study the role of AKT/Skp2/P27kip1 pathway in the prevention and treatment of HPH by estrogen.
(3) to explore the role and mechanism of plant estrogen in the prevention and treatment of HPH.
Experimental methods:
First, observe and explore the role and mechanism of endogenous estrogen in HPH.
We studied the level of the animal, pulmonary artery ring and pulmonary artery smooth muscle cells. First, the adult female, male, pregnant and ovariectomized SD rats (Sprague-Dawley rats) were randomly divided into 8 groups: the normal oxygen group and the hypoxic group. The atmospheric pressure in the normal oxygen group (the atmospheric pressure in Xi'an area was about 718mmHg, pO2 was 150.6m MHg), hypoxic group was placed in hypoxic chamber to decompress to 380mmHg (corresponding pO2 to 79.6mmHg, equivalent to oxygen partial pressure at altitude of 5000 m). The rat model of HPH was replicated for 21 days and the hemodynamic indexes of rats in each group were compared, such as right ventricular systolic pressure (Right ventricular systolic pressure, RVSP), mean pulmonary arterial pressure (mean pulmonar). Y arterial pressure, mPAP) and the pathological changes of the pulmonary artery. Secondly, the male and the female rats with different estrus cycles were taken to take the third stage of the pulmonary artery (300 m) of the pulmonary artery, and the vascular strips were immersed in the Krebs-Henseleit solution at 37 degrees C and 60 minutes under the condition of mixed gas (95%O2:5%CO2), and then replaced by the mixture of hypoxic oxygen. Gas (95%N2:5%O2), the estrogen receptor modulator raloxifene and the MAPK inhibitor U0126 (MAPK as the downstream pathway of the estrogen receptor GPR30) were intervened, and the endogenous estrogen levels were measured by radioimmunoassay, and the physiological changes of endogenous estrogen were observed in the pulmonary artery under the acute hypoxia condition. In the end, the rat pulmonary artery from different sex and estrus cycles was used to culture the smooth muscle cells in the primary culture, and cultured in 2% hypoxia conditions, using raloxifene and U0126 respectively. [3H]- thymidine deoxynucleoside incorporation was used to determine the proliferation of pulmonary artery smooth muscle cells induced by hypoxia, and then the endogenous female excitation was observed. Relationship between plasma level and proliferation of pulmonary artery smooth muscle cells in hypoxia.
Two, observe the role of exogenous estrogens in HPH and explore whether P27kip1 participates in its intrinsic protective mechanism.
On the basis of the replication of the HPH model of male rats, we also gave exogenous estrogen beta estradiol intervention to observe the hemodynamic parameters associated with HPH and the pathomorphological changes of the pulmonary arterioles. By immunoblotting experiments, the A related to cell cycle in pulmonary tissue and pulmonary artery smooth muscle cells was measured by reverse transcription PCR. KT/Skp2/P27kip1 and other key molecules, combined with the effect of beta estradiol on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia, explore the protective effect of exogenous estrogen on HPH by regulating the cell cycle at whole and cell levels.
Three, we observed and preliminarily explained the protective effect and mechanism of phytoestrogen resveratrol on HPH.
In order to find a compound with good effect and small side effect, we further observe the effect of phytoestrogen resveratrol on the prevention and control of HPH. At the overall level, we replicate the HPH model of male rats, and give the phytoestrogen resveratrol (40mg/kg/day, gavage), and also observe the hemodynamic parameters related to HPH and the disease of the pulmonary arterioles in rats. Morphological changes, immunohistochemical staining method was used to observe the infiltration of inflammatory cells around the pulmonary artery tissue in rats. Realtime-PCR was used to detect the expression of IL-6, TNF- alpha and other inflammatory mediators in the lung tissue, and the immunoblot test was used to observe the HIF-1 alpha, NF- kappa B, Nrf-2/Trx-1, H2O2, GSH, SOD, and so on. The changes in the expression of the key molecules; at the same time in the cell level hypoxic culture of pulmonary artery smooth muscle cells, different concentrations of phytoestrogen resveratrol were given, and estrogen receptor inhibitor ICI182780 was given to block estrogen receptor. WST-1 experiment was used to observe the effect of resveratrol on hypoxic pulmonary artery smooth muscle. In addition to the effect of cell proliferation, the inhibition of HIF-1 alpha by using HIF-1 alpha inhibitor KC7F2 and the inhibition of AKT pathway and ERK pathway with AKT and ERK inhibitors LY294002 and PD98059 respectively, the effects and mechanisms of phytoestrogen resveratrol on the proliferation of HPH and hypoxic pulmonary artery smooth muscle in rats were further revealed from the two aspects of inflammation and oxidative stress.
Experimental results:
1) the physiological changes of endogenous estrogen could affect the development of HPH in rats. The female rats with high estrogen level showed a lighter RVSP, mPAP and other hemodynamic indexes, and the thickening of the pulmonary artery wall in the rats with the high level of estrogen, while the HPH parameters of the female rats with the highest efemale level were the lightest. The estrogen level of the ovariectomized hypoxic rats was close to that of the male rats. The elevation of RVSP, mPAP and the thickening of the pulmonary artery wall were also close to the male hypoxia group. In vitro, the blood vessel test showed that the pulmonary arteries of the higher estrus rats were compared with the male rats and the estrous interval rats. The light HPV reaction and the MAPK inhibitor U0126 increased the HPV reaction. The cell level experiment further showed that the pulmonary artery smooth muscle cells from the estrous proestrus rats showed a lighter proliferation response. The intervention of raloxifene and U0126 enhanced the proliferation of pulmonary artery smooth muscle cells induced by hypoxia to some extent, and Ralo. When Xifen and U0126 intervened at the same time, they showed a superposition effect, which significantly increased the proliferation of pulmonary artery smooth muscle cells induced by hypoxia.
2) hypoxia leads to a significant increase in RVSP and right ventricular remodeling index RVHI, significantly thickening of the vascular wall of the pulmonary arterioles, and a significant decrease in the expression of the cell cycle inhibitory factor P27kip1 in the lung tissue, as well as the apparent increase in the expression of the P27kip1 specific degrading enzyme S kinase protein 2 (Skp-2) and the phosphorylated AKT protein. Sexual estrogen beta estradiol effectively alleviates the increase of RVSP and right ventricular remodeling index RVHI in rats induced by hypoxia, reduces the thickening of the vascular wall of the pulmonary arterioles, increases the expression of the phosphorylated AKT and Skp-2, and increases the expression of the cell cycle inhibitory factor P27kip1; the cell experiment validates the general experimental results and shows the beta estradiol. It can significantly reduce the proliferation of pulmonary artery smooth muscle cells induced by hypoxia.
3) phytoestrogen resveratrol obviously inhibits the development of HPH in rats, reduces hypoxic induced right ventricular pressure and right ventricular remodeling, reduces HIF-1 a caused by hypoxia, increases the expression of NF- kappa B in lung tissue, reduces the infiltration of pulmonary artery circumference in the pulmonary artery and the formation of H2O2 in the lung tissue, and reduces hypoxia. The increase of mRNA levels of inflammatory factors, such as IL-6, TNF- a, increases the expression of antioxidant stress factors such as Nrf-2, Trx-1, GSH and SOD, and the cell test shows that veratrol reduces the expression of HIF-1 a induced by hypoxia in the pulmonary artery smooth muscle cells in a concentration dependent manner, and inhibits the expression of HIF-1 alpha effectively. The proliferation of hypoxic pulmonary artery smooth muscle cells and the inhibitory effect of resveratrol inhibitor ICI182780 on the proliferation of hypoxic pulmonary vascular smooth muscle can not be blocked by resveratrol inhibitor. Resveratrol inhibits the proliferation of pulmonary artery smooth muscle cells induced by hypoxia and inhibits the expression of phosphorylated AKT and ERK.
Conclusion:
1) endogenous estrogen plays an antagonistic role in the development of rat HPH. It may reduce the proliferation of PVR and PASMCs by reducing the HPV and genomic pathways through the non genomic pathway (GPR30 receptor pathway) and thus exerts its antagonistic effect on HPH.
2) exogenous estrogen beta estradiol can effectively improve the HPH of rat, and it is one of the important mechanisms of inhibiting the proliferation of pulmonary artery smooth muscle cells by regulating the AKT/Skp2/P27kip1 pathway.
3) phytoestrogen resveratrol alleviates HPH, which is related to the inhibition of HIF-1 alpha pathway and the reduction of inflammatory response, as well as the activation of the Nrf-2/Trx-1 pathway, and the reduction of oxidative stress. The effect may be a non estrogen receptor dependent manner, but it is related to the inhibition of the AKT and ERK pathways.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R544.1

【引證文獻】

相關(guān)期刊論文 前1條

1 魏毅濤;段煉;王晉;劉曼玲;趙澎濤;;中草藥對低氧性肺動脈高壓的保護性作用、相關(guān)機制及研究進展[J];心臟雜志;2016年01期

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