【作者】 刘芳；

【导师】 格日力； 李占全；

【作者基本信息】 青海大学 ， 内科学， 2012， 博士

【摘要】 氧平衡状态的维持是需氧生物生存的根本前提。青藏高原环境的低氧独特性深刻影响着生物的分布、种群结构、生存适应和进化模式。藏羚羊(Tibetan antelope,Pantholops hodgsonii)是青藏高原土著动物中最具运动能力和低氧耐受能力的特有珍稀野生物种。该物种常年生活在海拔4300~5500m的高山草原、草甸和高寒荒漠地区，在生理、形态和基因水平上已获得稳定的低氧适应性遗传学特征，也形成了能够在低氧环境中生存的独特低氧适应机制。该机制的建立归结于机体内诸多抗低氧基因的表达水平发生了可调节性改变。在众多的抗低氧基因中，信号转导与转录激活因子3(signal transducer and activator of transcription3, STAT3)、低氧诱导因子-l(hypoxia inducible factor-l，HIF-l)、低氧诱导因子-2(hypoxia induciblefactor-2，HIF-2)是氧信号转导系统的核心转录因子，它们对于调节细胞低氧适应性反应、维持机体氧平衡状态和能量平衡状态起着关键性作用。STAT3是整合多种信号通路的关键性信号级联成分，具有信号转导与转录激活的功能。它可调控血管新生、能量代谢、细胞生长、存活与凋亡等途径中多种低氧反应性基因的表达，其中hif-1是STAT3的重要下游靶基因。HIF-1是关键性氧依赖转录激活因子。它通过诱导糖代谢、血管新生、红细胞生成、氧摄取及运输等途径中众多低氧反应性基因的表达，参与细胞内氧平衡状态的调节。HIF-2也是重要的氧依赖转录激活因子。同作为低氧诱导因子(hypoxia inducible factors,HIFs)家族成员，HIF-1和HIF-2均由氧调节亚基α和结构亚基β组成，α亚基是调节HIF-1、HIF-2活性的功能亚单位。HIF-1α、HIF-2α的氨基酸序列有48%的同源性，有很相似的结构域。HIF-1和HIF-2调节的下游靶基因既有重叠又各具特异性。HIF-2作用的靶基因主要涉及骨髓造血、血管生长、血管收缩、能量代谢、儿茶酚胺合成和铁代谢等方面。低氧条件下，HIF-1和HIF-2是以功能互补、协同作用的方式，促进细胞对低氧的适应调节，维持机体的氧平衡状态。可见，STAT3、HIF-1、HIF-2作为氧信号转导系统的重要转录因子，对于调节细胞低氧适应性反应起重要作用。我们推测上述三因子可能在藏羚羊的高原低氧适应机制中也扮演重要角色。但是藏羚羊stat3、hif-1α、hif-2α cDNA的克隆及组织表达特征鲜为报道，这一部分却是藏羚羊低氧适应分子机制研究中的重要内容，故本研究以高海拔藏羚羊和藏系绵羊及低海拔绵羊为材料分析了stat3、hif-1α、hif-2α的组织表达情况和低氧适应性差异，进而揭示藏羚羊在自然生活条件下stat3、hif-1α、hif-2α基因的表达特征及其对该物种生存适应的生理意义。本研究以高海拔藏羚羊、藏系绵羊和低海拔绵羊为材料，采用RT-PCR、3′，5′RACE PCR扩增分别获得藏羚羊stat3、hif-1α的cDNA克隆以及hif-2α的部分编码区序列，利用Real-time PCR和Western blot方法分析了藏羚羊、藏系绵羊和低海拔绵羊stat3、hif-1α、hif-2α的mRNA及蛋白的组织表达情况和低氧适应性差异。主要研究结果如下：1)序列分析结果显示，(a)藏羚羊stat3基因的cDNA序列为4288bp，包含2312bp的开放阅读框、29bp的5′UTR区和1947bp的3′UTR区。它的编码区序列和预测的氨基酸序列与其它哺乳动物stat3的相似性超过86%，其中与牛相似性最高，达到98%以上。(b)藏羚羊hif-1α基因的cDNA序列为3664bp，包含2471bp的开放阅读框和1191bp的3′UTR区，它的编码区序列和预测的氨基酸序列与其它哺乳动物hif-1α的相似性超过87%，其中与牛相似性最高，达到98%以上。(c)藏羚羊hif-2α基因的部分编码区序列为515bp，它的核苷酸序列与其它哺乳动物hif-2α的相似性为88%~97%左右，其中与牛的HIF-2α序列相似性最高。上述结果表明本实验获得藏羚羊stat3、hif-1α基因的cDNA序列和hif-2α基因的部分编码区序列。2) Real-time PCR和Western blot结果显示，(a)无论是mRNA水平还是蛋白水平，藏羚羊stat3、hif-1α、hif-2α在肺、肝、肾、心肌、骨骼肌组织中均有明显表达，且存在表达的组织差异性。与低海拔绵羊相比较，藏羚羊和藏系绵羊STAT3、HIF-1α、HIF-2α的蛋白表达量在上述五种组织中呈明显增高趋势。(b)以低海拔绵羊为对照，藏羚羊、藏系绵羊stat3、hif-1α、hif-2α mRNA的表达变化趋势并不一致。藏羚羊stat3、hif-1α、hif-2α mRNA在上述五种组织中的表达量均高于低海拔绵羊。藏系绵羊stat3、hif-1α、hif-2α mRNA的表达量在五种组织中的变化趋势各具特点，其中骨骼肌组织中各因子的mRNA表达量无明显差异。推测stat3、hif-1α、hif-2α的低氧特异性表达及表达增高可能是藏羚羊和藏系绵羊高原低氧适应能力和遗传学特征的分子基础。而同海拔藏羚羊和藏系绵羊之间所存在的stat3、hif-1α、hif-2α基因的表达差异性，则提示藏羚羊和藏系绵羊的低氧适应机制各不相同，它们依据各自的生存环境建立独特的低氧适应机制，这也进一步揭示藏羚羊，藏系绵羊在高原环境中遵从优胜劣汰、长期进化、适者生存的规律。更多还原

【Abstract】 The maintaining of oxygen homeostasis is the fundamental precondition ofaerobic organisms’ normal life activities. The hypoxic uniqueness of theQinghai-Tibet Plateau environment has a great impact on the distribution, structure,function, adaptation, and evolutionary patterns of various organisms. Tibetan antelope(Pantholops hodgsonii), is a rare wild species of the Qinghai-Tibet Plateau, whichhas the strongest sports ability and hypoxia resistant ability among aboriginal animalsof the Qinghai-Tibet Plateau. It perennially lives in alpine meadow, prata, alpine-colddesert areas at the altitude of4300~5500m. During long term evolutions, the specieshas already achieved genetic features of hypoxia adaptation in high altitude atphysiology, morphology and gene level, and has already formed unique adaptationmechanisms. The establishment of the adaptation mechanism due to expression levelsof hypoxia-adaptive genes can be adjusted to change. In a large number ofhypoxia-adaptive genes, Signal transduction and activator of transcription factor3(STAT3), Hypoxia inducible factor-1(HIF-1), Hypoxia inducible factor-2(HIF-2) isthe core transcription factors of the oxygen signal transduction system. They play akey role in regulating cell hypoxia adaptive response to maintain body oxygenequilibrium and energy balance status.STAT3is the key signaling cascade components which integrate signals ofmultiple signaling pathways, having functions of signal transducing and transcriptioncontrolling. It regulate gene expressions of hypoxia response in various ways, such ascell proliferation, survival and apoptosis, angiogenesis, energy metabolism, whichhif-1is an important downstream target genes of STAT3. As a criticaloxygen-dependent transcriptional activator, HIF-1through the induction of glucosemetabolism, angiogenesis, erythropoiesis, many of hypoxic response gene expressionin oxygen uptake and transport pathway involved in the regulation of the intracellularoxygen equilibrium. HIF-2is also a critical oxygen-dependent transcriptionalactivator. With family members as hypoxia inducible factors (HIFs)，HIF-1and HIF-2are compose of the oxygen regulation of subunit alpha and the structure of subunitbeta. Alpha subunit is the regulation of HIF-1and HIF-2activity. HIF-1α, HIF-2α,48%of the amino acid sequence homology, have very similar structural domains.HIF-1and HIF-2regulation of downstream target genes both overlap and each withspecificity. HIF-2target genes that mainly involves the bone marrow, blood vessel growth, vascular contraction, energy metabolism, catecholamine synthesis and ironmetabolism. Under hypoxic conditions, HIF-1and HIF-2are in a functioncomplementary, synergistic manner that promote cell adaptation to hypoxia regulateand maintain the body′s oxygen equilibrium. In summary, as important transcriptionfactors of the oxygen signal transduction system, STAT3, HIF-1and HIF-2play animportant role in the regulation of cell hypoxia adaptive responses. We speculate thatSTAT3, HIF-1and HIF-2plays a very important role in Tibetan antelopes′adaptionmechanisms to hypoxia. But there are few reports on the cloning and tissue expressioncharacters of stat3,hif-1α and hif-2α cDNA, which are the important content of theresearch on the hypoxia adaption molecular mechanism of Tibetan antelope. So thisresearch analyzed the differences of the tissue expression and hypoxia adaption ofstat3,hif-1α and hif-2α, with Tibetan antelope, Tibetan sheep and sheep asmaterials, further uncovered the meaning of these genes expression character in natureand the physiological significance of stat3,hif-1α and hif-2α for Tibetan antelope tosurvive.In this study, the cloning of stat3,hif-1α and hif-2α gene cDNA of Tibetanantelope, using RT-PCR and RACE, was applied, as well as the comparative analysisof the tissue-specific expressions of stat3,hif-1α and hif-2α among Tibetan antelope,Tibetan sheep and sheep(Ovis aries), using Real-time PCR and Western blot.Themain achievement of this study is listed as follows:1)Sequence analysis revealed that (a)The full-length Tibetan antelope stat3cDNA,which cloned by gene sequence analysis, is4288bp, comprising a2312bp openreading frame (ORF), a29bp5′UTR and a1947bp3′UTR. The similarity between itscoding sequence, predicted amino acid sequence and stat3of other mammalsexceeded86%, in which the similarity with cow was up to more than98%.(b)ThecDNA sequences acquired by cloning from the hif-1α gene of Tibetan antelopecomprised a2471bp ORF and a1911bp3′UTR. The similarity between its codingsequence, predicted amino acid sequence and hif-1α of other mammals exceeded87%,in which the similarity with cow was up to more than98%.(c)Partial coding regionsequence of Tibetan antelope′s hif-2α gene is515bp. Similarity between its codingsequence and hif-2α of other mammals is about88%~97%, in which the similaritywith cow is the highest. The results indicate that cDNA sequences of Tibetanantelope′s stat3and hif-1α and partial coding region sequence of Tibetan antelope′shif-2α gene were obtained. 2)Real-time PCR and Western blot analysis revealed that (a)Both the mRNA level orprotein level, stat3,hif-1α and hif-2α of Tibetan antelope′s were significantlyexpressed in the lung, liver, kidney, cardiac muscle, skeletal muscle tissue and thereare organizational differences in expression. The compare of the same tissues amongthe three kind of animals shows that the expression of STAT3, HIF-1α and HIF-2αprotein in the five tissues of Tibetan antelope and Tibetan sheep are all more than thatof sheep.(b)Sheep as control, there were not consistent between the expression ofstat3, hif-1α and hif-2α mRNA in the five tissues of Tibetan antelope and them ofTibetan sheep.The expression of stat3,hif-1α and hif-2α mRNA in the five tissues ofTibetan antelope are all more than that of sheep. However, there were variouscharacteristics about the expression of stat3,hif-1α and hif-2α mRNA in the fivetissues of Tibetan sheep, in which expression of these factors′mRNA was nosignificant difference in skeletal muscle. We speculated that the tissue specificexpression and higher expression of stat3,hif-1α and hif-2α is molecular basis ofTibetan antelope and Tibetan sheep′s adaptability and genetic features. The expressionof stat3,hif-1α and hif-2α gene differences between Tibetan antelope and Tibetansheep, although they live in the same altitude. It indicates Tibetan antelope andTibetan sheep establish their own unique hypoxia adaptation mechanisms based ontheir living environment. It further announced that Tibetan antelope and Tibetan sheepcomply with the laws of optimizing, long-term evolution and survival of the fittest inthe highland environment.更多还原