缺氧诱导因子1α和诱导型一氧化氮合酶基因在缺氧性肺动脉高压大鼠发病过程中的表达(英文)

Expressions of hypoxia inducible factor-1 alpha and inducible nitric oxide synthase gene in the development of hypoxic pulmonary hypertension in rats

背景:关于缺氧性肺动脉高压发病过程中缺氧诱导因子1α和诱导型一氧化氮合酶在肺动脉壁内的动态变化尚需探讨。目的:观察不同缺氧时间点肺动脉壁内缺氧诱导因子1α和诱导型一氧化氮合酶基因表达,探讨其在缺氧性肺动脉高压发病机制中的作用。设计:对比观察的动物实验。单位:南华大学附属第二医院呼吸内科。材料:选用健康雄性Wistar大鼠40只,清洁级,6～8周龄,体质量(220±10)g。按随机表法分为对照组(n=8)和缺氧组(n=32),其中缺氧组又分为缺氧后3,7,14,21d4个时间点进行观察,每个时间点8只。方法:实验于2004-08/2005-12在南华大学肿瘤研究所完成。缺氧组大鼠按李启芳报道的方法进行干预。对照组大鼠置于同一室内,除不缺氧外,余同缺氧组。按照观察时间点将大鼠麻醉后,右颈外静脉插入微导管,连接多导生理记录仪,检测大鼠肺动脉平均压。将大鼠处死取出心脏称量右心室、左室加室间隔的质量,以右室肥大指数反映右心室肥厚程度。取大鼠右上肺组织,进行苏木精-伊红染色和弹性纤维染色。用病理图像分析软件测定肺动脉管壁面积/管总面积、管腔面积/管总面积、肺细小动脉中膜平滑肌细胞密度、肺细小动脉中膜厚度作为肺小血管重塑指标。对肺小血管壁缺氧诱导因子1α,诱导型一氧化氮合酶进行原位杂交和免疫组织化学检测,以肺细小动脉管壁平均吸光度值作为mRNA表达和蛋白水平的相对含量。主要观察指标:①大鼠肺动脉平均压、右心室肥厚程度和肺小血管重塑指标的变化。②缺氧诱导因子1和诱导型一氧化氮合酶的表达及其与肺动脉平均压,肺血管重塑的关系。结果:共纳入Wistar大鼠40只全部进入结果分析。①缺氧7d时肺动脉平均压明显高于对照组(P<0.05),14d达到最高水平,之后维持于此水平。②缺氧14d右心室肥大指数高于对照组(P<0.05)。③缺氧7d肺小动脉管壁增厚,管腔变窄,肺动脉管壁面积/管总面积、管腔面积/管总面积与对照组比较,差异明显(P<0.05);缺氧14d可见肺小动脉中膜和肺细小动脉中膜平滑肌细胞密度增高,肺细小动脉中膜厚度明显增加(P<0.05)。21d管腔进一步变窄,平滑肌增生明显。④缺氧诱导因子1和诱导型一氧化氮合酶mRNA的表达:对照组大鼠呈弱阳性表达;缺氧3d和7d缺氧诱导因子1αmRNA相对量无明显变化,14d时明显增高,此后维持于高水平。诱导型一氧化氮合酶mRNA在缺氧3d明显高于对照组,7d达到高峰,14d接近对照组水平,21d再次升高,但低于缺氧3d时。⑤缺氧诱导因子1α主要表达于血管内膜和中膜,而诱导型一氧化氮合酶表达涉及血管全层。诱导型一氧化氮合酶在对照组肺血管内膜和中膜均弱阳性表达,缺氧3d与对照组差异不明显,7d中膜、内膜表达明显,14d血管中膜增厚,表达增强。在血管外膜,对照组诱导型一氧化氮合酶为阴性,各缺氧组均阳性表达。⑥mPAP与肺血管重塑正相关(r=0.976,P<0.01),缺氧诱导因子1αmRNA与诱导型一氧化氮合酶蛋白正相关(r=0.927,P<0.05)。结论:缺氧诱导因子1α和诱导型一氧化氮合酶均在大鼠缺氧性肺动脉高压的发病过程中发挥作用,且缺氧诱导因子1α与诱导型一氧化氮合酶基因表达可能存在相互调控。

BACKGROUND：The dynamic changes of hypoxia inducible factor-1 alpha （HIF-α） and inducible nitric oxide synthase （iNOS） genes in the pulmonary artery wall during the process of hypoxic pulmonary hypertension （HPH） development need to be investigated. OBJECTIVE： This study was to observe the gene expressions of HIF-α and iNOS in the pulmonary artery wall at the different hypoxic time points, and to investigate their effects in HPH development. DESTGN： Controlled observation animal experiment SETTING： Department of Respiration, Second Hospital Affiliated to Nanhua University MATERIALS： Forty healthy male Wistar rats of clean grade, aged 6-8 weeks, with body mass of （220±10） g were involved in this study. They were randomized into control group （n =8） and hypoxia group （n =32）. Four time points, i.e. 3, 7, 14, and 21 days after hypoxia were set for the animals in the hypoxia group, 8 rats at each time point. METHODS： This study was carried out in the Institute of Oncology, Nanhua University between August 2004 and December 2005. Rats in the hypoixa group were treated according to the method reported by Li et al. Hypoxia treatment was omitted for the rats in the control group. At each time point, the rats were anesthetized, and then a micro-catheter was inserted into the right jugular vein and connected to a multichannel physiologic recorder, which was used for detecting the mean pulmonary arterial pressure （mPAP）. The heart of each euthanized rat was taken out, and its right ventricle （RV）, and left ventricle and septum （LV＋S） were weighted. Right ventrical hypertrophy index （RVHI） reflected right ventricle hypertrophy degree. Right upper lung tissue of rat was harvested for haematoxylin ＆ eosin （HE） staining and elastic fiber staining. Pathological image analysis software was used to determine pulmonary arterial wall area/total vascular area, lumina areal total vascular area, smooth muscle cell density in the media of pulmonary arteriole, and media thickness of pulmonary arteriole, which were used as remodeling indexes of pulmonary arteriole. HIF-1α and iNOS in the pulmonary arteriole were performed in situ hybridization and immunohistochemical detection. The mean absorbance of pulmonary arteriole wall was used as the relative content of mRNA expression and protein level. MAIN OUTCONE MEASURES： ①Changes in mPAP, RV arteriole of rats. ②Expressions of HIF-α and iNOS as we pulmonary arteriole. hypertrophy degree and remodeling indexes of pulmonary as their correlations with mPAP and remodeling indexes of RESULTS ： All the 40 Wistar rats were involved in the final analysis.①At hypoxia 7 days, mPAP was significantly higher than that of control group （P 〈 0.05）. mPAP reached to the high level at hypoxia 14 days, and then maintained at this level. ②At hypoxia 14 days, RVHI was higher than that of control group （P 〈 0.05）. ③At hypoxia 7 days, pulmonary arteriole wall was thickened, and lumina became narrowed. There were significant differences in pulmonary arterial wall area/total vascular area, lumina areal total vascular area between hypoxia group and control group （P 〈 0.05）. At hypoxia 14 days, smooth muscle cell density in the intima-media of pulmonary arteriole, and intima-media thickness of pulmonary arteriole were significantly increased （P 〈 0.05）. At hypoxia 21 days, lumina was further narrowed, and obvious smooth muscle hyperplasy was found. HIF-1α and iNOS mRNA expression presented weak-positive in the control group; The relative content of HIF-1α mRNA did not significantly alter at hypoxia 3 and 7 days, but was obviously increased at hypoxia 14 days ,and after this, it maintained at high level, iNOS mRNA was markedly higher than that in the control group at hypoxia 3 days, reached its peak at hypoxia 7 days, was close to the level in the control group at hypoxia 14 days, and enhanced again at hypoxia 21 days, but it was still lower than that at hypoxia 3 days. ⑤HIF-α was mainly found in the intima and media, while iNOS was found in the whole layer of vessels, iNOS was weakly expressed in the intima and media of pulmonary vessels in the control group. No obvious difference in iNOS existed at hypoxia 3 days as compared with control group, iNOS was obviously expressed in media and intima at hypoxia 7 days. Vascular media was thickened at hypoxia 14 days, and the expression of iNOS was enhanced, iNOS was not found in the vascular adventitia in the control group, but was found in the vascular adventitia in the hypoxia group. ⑥mPAP was positively correlated with pulmonary vascular remodeling （r =0.976, P 〈 0.01）, and HIF-1α mRNA was positively correlated with iNOS protein（r = 0.927, P 〈 0.05）. CONCLUSION ： Both HIF-1α and iNOS exert effects in the process of HPH development of rats, and HIF-1α and iNOS gene expression may be mutually regulated.