诱导型一氧化氮合酶（iNOS）在大通牦牛肺组织低氧适应中的作用

In this work, Datong yaks, as research objects, were compared with yak of around-lake type, acclimatization cattle and plain cattle from the region at same attitude. By using the techniques such as comparative physiology, molecular biology, the full-length cDNA of the inducible NO-synthase (iNOS) were obtained, the enzymatic activities, the expression profiles and the promoter activities of the iNOS were analysed and compared in different groups. On this basis, the responses of the iNOS of the Datong yaks under hypoxic environment were analyzed. This work aimed to determine the loci of adaptive evolution and hypoxic response elements of the iNOS for the Datong yaks, and discuss the relationships between the molecular evolution and expression regulation of the iNOS for the Datong yaks, and hypobaric hypoxia environment. By doing so, the authors can reveal the effect and mechanism of the molecular evolution and expression regulation of the iNOS on the hypoxic adaptation of the pulmonary tissues for the Datong yaks. This research is expected to provide theoretical and experimental basis for preventing and treating high-altitude diseases such as the pulmonary hypertension and pulmonary edema by using iNOS.

本项目选取“大通牦牛”作为研究对象，并以同海拔环湖型牦牛、高原习服黄牛和平原黄牛作对照，采用比较生理学、分子生物学等技术手段，获得“大通牦牛”iNOS的全长cDNA序列；比较各组试验动物iNOS酶活、iNOS表达谱和启动子活性等的差异；分析“大通牦牛”iNOS对低氧的应答。其目的在于确定“大通牦牛”iNOS基因的适应性进化位点和低氧反应元件，探讨“大通牦牛”iNOS分子进化、表达调控与高原低氧压力环境之间的关系，以揭示iNOS的分子进化及表达调控在“大通牦牛”肺循环低氧适应中的作用与机制，为利用iNOS防治肺动脉高压、肺水肿等高原病提供理论和实验依据。

为阐明诱导型一氧化氮合酶在高原牦牛肺组织低氧适应中的作用，本实验选择不同海拔的高原牦牛和黄牛作为研究对象。颈动脉采血检测血液中NO水平，利用实时荧光定量PCR和蛋白免疫印迹方法对高原牦牛和黄牛肺组织中iNOS mRNA及蛋白的相对表达量进行分析。并构建大通牦牛iNOS-p3×FLAG-CMV-10表达载体，转染HEK293T细胞,以p3×FLAG-CMV-10空质粒为对照，利用Western Blot及ELISA技术进行体外表达及酶活性检测，研究结果显示： .（1）黄牛血液中NO含量随海拔增加呈递增趋势，海晏黄牛血液中NO含量最高，显著高于同海拔的海晏牦牛；大通牦牛血液中NO含量高于同海拔海晏牦牛，但差异不显著。.（2）黄牛和牦牛肺组织中iNOS mRNA相对表达量均呈现海拔递增趋势，海晏黄牛最高；急进高原秦川黄牛显著高于秦川黄牛；大通牦牛高于同海拔海晏牦牛，但差异不显著。.（3）黄牛和牦牛肺组织中iNOS蛋白相对表达量呈现海拔递增趋势，海晏黄牛显著高于海晏牦牛；急进高原秦川黄牛显著高于秦川黄牛；大通牦牛显著高于海晏牦牛。.（4）成功构建牦牛iNOS-p3×FLAG-CMV-10表达载体，转染HEK293T细胞后，iNOS蛋白及酶活性显著高于空质粒转染组，iNOS-p3×FLAG-CMV-10表达载体可瞬时、有效表达。.以上结果表明肺组织中iNOS mRNA、蛋白表达和其血液中NO含量趋势具有一致性，表明iNOS的表达调控主要发生在转录水平；肺组织iNOS mRNA、蛋白表达和血液中NO含量受到海拔高度和品种的共同影响；肺组织中iNOS的表达在动物适应低氧环境过程中发挥一定作用。.