NWIPB OpenIR
家牦牛低氧诱导因子-1α (HIF-1α)基因的表达特征及其低氧适应意义
其他题名Characteristic of Hypoxia Inducible Factor-1α Gene Expression and Its Significance of Hypoxic Adaptation in Domestic Yak
王德朋
学位类型博士
导师赵新全
2007-06-09
学位授予单位中国科学院西北高原生物研究所
学位授予地点西北高原生物研究所
关键词青藏高原 牦牛低氧适应 Hif-1α Epo Nos
摘要青藏高原又被称为“地球的第三极”,高海拔、缺氧、严寒和强辐射是它的主要特征。低氧是阻止动物生存的最重要的生态因子,在长期的进化过程中,高原土著动物形成了一套它们独特的机制来适应高原的缺氧环境。牦牛是青藏高原上大型草食动物,在青藏高原的进化历史至少可追朔到二百万年前。牦牛经过长期的自然选择,在生理、生化和形态学上已获得了稳定的适应高原低氧的遗传学特征,如没有肺动脉高压和右心肥厚,心肺发育良好,心肺指数高等,但它对低氧适应的分子机制目前还不清楚。HIF-1是一个转录激活因子,主要调控组织和器官的氧的动态平衡,它是一个由两个亚单位(HIF-1α和HIF-1β)组成的异二聚体。HIF-1的调控活性主要涉及α亚单位,发生在多个水平,比如蛋白的稳定、翻译后的修饰、核内定位、二聚化、转录激活以及与其他蛋白的相互作用。 本研究选择青海家养牦牛作为研究对象,通过RT-PCR得到了两个不同的HIF-1α完整开放阅读框。进一步分析发现,两个不同的HIF-1α完整开放阅读框翻译的氨基酸序列有高度的保守性。发现和鉴定了一个新的选择性剪切的mRNA转录的HIF-1α异构体,它在5’端多出了并不在bHLH区的123个核苷酸,编码41个氨基酸,它可能在牦牛的低氧适应方面有着积极的作用。使用Northern Blot和Real Time RT-PCR,检测了HIF-1α mRNA在牦牛八种组织(心、肝、脾、肺、肾、脑、肌肉、睾丸)中的表达;使用Western Blot检测了HIF-1α蛋白在牦牛这八种组织中的表达。结果表明,牦牛HIF-1α mRNA和牦牛HIF-1α蛋白均为组织特异性表达,但二者的表达量在同一个组织中并不是完全一致的。牦牛HIF-1α mRNA主要在睾丸和脾中高表达,而牦牛HIF-1α蛋白的高表达主要在脑、肺、肾和脾中。牦牛HIF-1α mRNA与HIF-1α蛋白组织特异性表达的不一致,提示我们HIF-1α可能是某些基因的靶基因,有其他因素而不只是低氧维持了HIF-1α mRNA稳定状态水平的增加。 HIF-1α蛋白在牦牛肾中的高表达,可能参与调控EPO的释放,以刺激骨髓产生更多的红细胞,更多的红细胞数量和更高的血红蛋白浓度是大多数高海拔动物的血液学的主要特征,这增强了血液运输氧的能力。EPO也被认为是一个多功能的生长因子在神经系统中起着明显的作用,脑源性EPO具有神经营养作用,是抵抗缺氧缺血性脑损伤的一个重要的内源性保护因子。EPO在脑的神经胶质细胞、神经元和内皮细胞中广泛表达,低氧已经被认为是促进EPO在脑中表达的主要的推动力量,而EPO又是HIF-1α下游调控的靶基因之一,HIF-1α蛋白在牦牛脑中的高表达可能正是起了这样的作用。生活在低海拔地区的动物来到高海拔地区之后,通常会出现肺动脉高压,并伴随着肺动脉的肌肉变的强壮。肺动脉高压的变化程度与在高海拔地区待的时间长短和低氧刺激的程度有关系。高海拔地区的牦牛与同种的牛相比已经成功的适应了低氧,因为当牛暴露在慢性低氧环境时,肺血管强烈收缩,动脉血管肌肉化,形成了严重的肺动脉高压。牦牛的小动脉血管厚度平均,没有平滑肌,与牛相比,牦牛的肺动脉内皮细胞更长、更宽、更圆,牦牛通过维持对低氧不敏感的肺动脉收缩和管壁较薄的肺血管,来成功适应高海拔。内源性NO是通过NO合成酶将保守的L-精氨酸酶解成L-瓜氨酸时产生的,NO通过抑制平滑肌的增殖、分化、发炎和大量蛋白的产生来维持系统和肺血管的舒张。因此肺部产生的NO增多能够改变血管的结构和阻止血管重建来抑制肺动脉高压的形成。因此我们推测HIF-1α蛋白在牦牛肺中的高表达能够增高NOS的表达以产生更多的NO,来阻止牦牛肺动脉高压的形成。 我们的研究表明HIF-1α在牦牛的低氧适应中起着非常关键的作用,但许多细节问题仍未了解清楚,需要以后的实验来进一步发现和证实。
其他摘要Qinghai-Tibetan Plateau is known as ‘the third pole of the earth’. High altitude, hypoxia, extreme cold and high solar radiation are its most dominant characteristics. Hypoxia is the most important ecological factor to inhibit survival of animals. Therefore, in long-term evolution, native plateau animals have formed their unique mechanism in adaptation to extreme plateau environment. The yak is the biggest animal which had lived on the Qinghai-Tibetan for at least two millions of years. Through perennial natural selection, it had gotten steady genetic characteristic which adapted to high altitude hypoxia on physiology, biochemistry and morphology such as no pulmonary hypertension and no evidence of right ventricular hypertrophy, having good development of heart and lung, and high cardiopulmonary exponent. But the molecular mechanism of the yak how to adapt the hypoxia environment is known indistinctly. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator that functions as a master regulator of cellular and systemic oxygen homeostasis. Hypoxia-inducible factor-1 is a heterodimer composed of the α and β subunits. The regulation of HIF-1 activity concerns mostly the α subunit and occurs at multiple levels such as protein stabilization, posttranslational modifications, nuclear translocation, dimerization, transcriptional activation, and interaction with other proteins. In this study, the Qinghai domestic yak was selected as the model and we cloned two different HIF1-α ORFs (open reading frames) of domestic yak through the method of RT-PCR. Comparison of the different HIF1-α ORFs showed that the predicted amino acid sequences show high level of conservation. We identified and characterized a new alternatively spliced variant of HIF1-α ORF which had excessive 123 base pairs in the 5’ terminal and it can translate 41 amino acids. But the incremental 41 amino acids are not on the bHLH domain. They may play positive function in the adaptation to the hypoxia of the domestic yak. We did its mRNA expression in eight different tissues (heart, liver, spleen, lung, kidney, brain, muscle, testis) using the methods of Northern Blot and Real Time RT-PCR. We also did its protein expression in the same eight different tissues using the method of Western Blot. Data shown both HIF-1α mRNA and HIF-1α protein of the yak are tissue specific expression, but their expressive quantities in the same tissue are not completely consistent. In contrast its expression of HIF-1α mRNA with other animals, it highly expressed in the testis and spleen, but its HIF-1α protein highly expressed in the brain, lung, kidney and spleen. This informs that HIF-1α may be the target gene of other genes and factors other than hypoxia are necessary for the increase in HIF-1α mRNA. HIF-1α protein high expression in the kidney of the yak my particulate in the regulating the release of EPO, thus it can stimulate the bone marrow to produce more red blood cells. More red blood cells and higher concentration of the hemoglobin are the crucial characters of most high altitude animals, this enhances the capacity in transporting the oxygen of the blood. Also EPO is regarded as the multifunctional growth factor that it plays evident role in the neural system. Hypoxia-induced EPO appears to act directly on forebrain neural stem cells, promoting the production of neuronal progenitors and suggesting that EPO is involved in neurogenesis after hypoxia. Brain-derived EPO possessed trophic activity on the nerves and it can act on neurons as a neurotrophic factor, and it is an important endogenetic protective factor to resist with the hypoxic-ischemic brain damage. EPO is widely expressed in the astrocytes, neurons and endothelial cells in the brain, hypoxia has been regarded as the crucial power to promote EPO expression in the brain. And also EPO is one of the downstream regulating genes of HIF-1α, and HIF-1α protein high expression in the brain may play such role. It is well known that when mammalian species that live at sea level are exposed to high altitude, certain species develop pulmonary hypertension with increased muscularization of the pulmonary arterioles. The degree of pulmonary hypertension varies among species and is related to the duration of high altitude residence and magnitude of the hypoxic stimulus. The high altitude native yak has successfully adapted to chronic hypoxia despite being in the same genus as domestic cows, which known for their great hypoxic pulmonary vasoconstrictor responses, muscular pulmonary arteries, and development of severe pulmonary hypertension on exposure to chronic hypoxia. Yak small pulmonary arteries had medial thickness, with vessels devoid of smooth muscle. Yak pulmonary artery endothelial cells were much longer, wider, and rounder in appearance than those of domestic cows. Thus the yak has successfully adapted to high altitude conditions by maintaining both a blunted hypoxic pulmonary vasoconstrictor response and thin-walled pulmonary vessels. Endogenous NO is enzymatically produced from conversion of the amino acid L-arginine to L-citrulline, a reaction catalyzed by the enzyme NO synthase (NOS). NO is a potent systemic and pulmonary vasodilator that inhibits smooth muscle cell proliferation, migration and inflammation and matrix protein production. Hence enhancement in NO production in the lung may inhibit with the development of pulmonary hypertension by both decreasing vascular tone and preventing vascular remodeling. We speculate in the domestic yak lung that HIF-1α protein high expression increases NOS expression thus can produce more NO. Thus NO prevents from the formation of the pulmonary hypertension. We thought this is the main reason that HIF-1α protein highly expressed in the lung. Our research informs that HIF-1α protein plays crucial role in the adaptation to the hypoxia environment of the domestic yak, but there are many problems not to be understood clearly and this need later experiment to further find and prove.
页数127
语种中文
文献类型学位论文
条目标识符http://210.75.249.4/handle/363003/3102
专题中国科学院西北高原生物研究所
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GB/T 7714
王德朋. 家牦牛低氧诱导因子-1α (HIF-1α)基因的表达特征及其低氧适应意义[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2007.
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