高原鼠兔HIF-1α和VEGFs的表达特征及其在低氧适应中的作用

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	高原鼠兔HIF-1α和VEGFs的表达特征及其在低氧适应中的作用
其他题名	Expression patterns of HIF-1α and VEGF and their roles in hypoxic adaptation of the plateau pika
	李红阁
学位类型	博士
导师	赵新全
	2009-01-15
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	低氧适应青藏高原高原鼠兔低氧诱导因子-1α 血管内皮生长因子
摘要	高原鼠兔（Ochotona curzoniae）是生活在青藏高原海拔3000-5000 m区域的优势物种，具有高的低温、低氧耐受能力，是研究低氧适应机制的最佳动物模型。低氧诱导因子-1（Hypoxia Inducible Factor 1，HIF-1）是一个重要的转录因子，在细胞的和系统的低氧反应过程中起关键作用，其生物活性主要取决于氧调节亚基HIF-1α蛋白的稳定性和转录活性。血管内皮生长因子（Vascular Endothelial Growth Factor, VEGF）在组织低氧反应中是一个促进新血管生成的关键调节子，并且在血管舒张反应中具有重要的作用。VEGF在低氧条件下的诱导主要受HIF-1的调控。在本研究中，我们通过生物化学和分子生物学的方法以HIF-1α蛋白和VEGF为切入点研究了高原鼠兔高海拔低氧适应的分子机理。主要研究结果如下： 1.高原鼠兔HIF-1α蛋白的在生理环境中的实际大小为114KD，其主要定位到细胞核中，能够转录激活低氧反应元件（hypoxia response element, HRE）驱动的基因的表达，其蛋白的稳定性受HIF脯氨酸羟化酶羟基化的调控。这些结果与人和其它物种的HIF-1α蛋白的研究相一致，提示了鼠兔HIF-1α蛋白功能特性的保守性。 2.制备了高原鼠兔HIF-1α蛋白的多克隆抗体，其能特异地识别鼠兔HIF-1α蛋白，也能识别人、小鼠和猴HIF-1α蛋白。 3.在高原鼠兔组织内可以明显检测到HIF-1α蛋白的表达，其表达水平高于平原小鼠，尤其是在肺、肝、脾和肾组织达到了显著水平。 4.随着生活海拔的升高，高原鼠兔肺、肾、肝和脾组织内HIF-1α蛋白水平增加。而海拔3200、3950和4750 m区域的高原鼠兔心脏和肌肉组织内HIF-1α mRNA水平并无明显差异。 5.高原鼠兔VEGF165和VEGF189全长编码区分别由576和648bp组成，分别编码由191和215个氨基酸组成的蛋白质。预测的蛋白分子量分别为19.43KD和22.32KD，等电点分别为7.93和9.27。 6.高原鼠兔VEGF165编码区核苷酸序列与人、兔、小鼠和挪威鼠的同源性分别为95、94、91和90% (VEGF189: 95、95、91和90%)。氨基酸序列同源性达89～96%。 7.高原鼠兔VEGF165和VEGF189蛋白由两个功能域组成：N端的受体结合结构域和C端的肝素结合结构域。预测的两个功能域的二级和三级结构分别与其它物种VEGF165的相应功能域相似。 8.系统进化分析表明，高原鼠兔VEGF165和VEGF189是高度保守的，可以作为分子标记来揭示分类学中的系统发生关系。 9.高原鼠兔总的VEGF以及VEGF165和VEGF189mRNA的表达具有组织特异性。有意思的是，VEGF165和VEGF189在高原鼠兔脑和肌肉组织内的表达模式与平原小鼠相反。随着生活海拔的升高，高原鼠兔脑、肌肉和肺组织内VEGF189的表达升高，而VEGF165表达无明显变化或比VEGF189的变化少。 10.高原鼠兔肺组织内HIF-1α蛋白和VEGF mRNA海拔相关的变化趋势相似，相关分析表明二者存在明显的正相关。我们的研究结果表明高原鼠兔HIF-1α蛋白和VEGF(尤其是VEGF189 ) mRNA可能在高原鼠兔低氧适应中具有重要的作用。
其他摘要	The plateau pika(Ochotona curzonia), a dominant species living at 3000-5000 m above sea level on the Qinghai-Tibetan plateau, is a good model for studying the mechanism of high-altitude hypoxia adaptation. Hypoxia- inducible factor-1 (HIF-1) is a key transcription factor that regulates a variety of cellular and systemic adaptations to hypoxia. Its activity is mainly regulated through oxygen-dependent regulation of HIF-1α protein stability and transcriptional activity. VEGF, vascular endothelial growth factor, has been proved to be a key regulator of angiogenesis in response to tissue hypoxia and plays an important role in vascular vasodilation. Hypoxia-induced expression of VEGF is mainly regulated by HIF-1. In this study, we study the molecular mechanism of high-altitude hypoxia adaptation by HIF-1α protein and VEGF through biochemical and molecular techniques. The results are presented as follows: 1. The molecular weight of plateau pika (p) HIF-1α protein is 114 KD under the physiological condition. It is mainly localized to the cellular nucleus and has potential to transactivate the expression of a HRE-driven gene. The stability of pika HIF-1α protein is regulated through the prolyl hydroxylation catalyzed by PHDs (HIF prolyl hydroxylases). The expression, localization, function and regulation of pika HIF-1α protein are similar with those of other species’ HIF-1α. 2. We generated the polyclonal anti-HIF-1α antibody with pika HIF-1α protein antigen, which specifically recognized pHIF-1α. In addition, this antibody also recognized human, mouse and monkey HIF-1α protein. 3. HIF-1α protein was expressed at a significantly high level in the plateau pika. The HIF-1α protein remained at high levels in most of the tissues in the pika comparing with that in the sea-level mouse. Especially, HIF-1α protein level was found to be elevated in the lung, liver, spleen and kidney of the pika. 4. The protein levels of HIF-1α in the lung, liver, spleen and kidney of the pika were increased with the increased habitat altitudes. However, the HIF-1α mRNA expression levels in the heart and muscle show no distinctive differences between the pikas at 3200, 3950 and 4750 m. 5. The full-length coding sequences of plateau pika VEGF165 and VEGF189 consist of 576 and 648 bp, respectively. The predicted VEGF proteins from these isoforms were 191 and 215 amino acids in length. The mature excreted protein has a predicted molecular weight of 19.43 KD and 22.32 KD and a pI of 7.93 and 9.27, respectively. 6. Plateau pika VEGF165 shares 95, 94, 91 and 90% of nucleotide sequence homology to the orthologs in human, rabbit, mouse and Norway rat. The similarity of pika VEGF189 to the orthologs in human, rabbit, mouse and Norway rat is also high (95, 95, 91 and 91%, respectively). A multiple sequence alignment revealed that the pVEGF165 and pVEGF189 proteins were 89-96% identical to those of other mammals. 7. Plateau pika VEGF165 or VEGF189 is predicted to consist of a receptor-binding domain and a heparin-binding domain. The predicted secondary and tertiary structures of the two VEGF domains have high similarity between the pika and other species. 8. The phylogenetic analysis further reveals that VEGF165 and VEGF189 are evolutionarily conserved. Therefore, the VEGF gene may be a good phylogenetic performer in recovering the true phylogenetic relationships among taxa. 9. Total VEGF and VEGF165 and VEGF189 mRNAs display tissue and altitude-specific expression patterns. Interestingly, VEGF165 and VEGF189 mRNAs display a reversed expression pattern in the brain and muscle in the pika compared with the sea-level mouse. Futher, VEGF189 mRNA levels in the brain, muscle and lung of the pika increased along with increasing habitat altitude, while VEGF165 showed no difference or less difference than VEGF189 between altitudes. 10. The trends of changes in HIF-1α protein and VEGF mRNA expression along with altitudes are similar. And there is a high positive correlation between HIF-1α protein and VEGF mRNA expression. In conclusion all the results indicate that HIF-1α protein and VEGF (especially VEGF189) play an important role in the high-altitude hypoxia adaptation of the plateau pika.
页数	112
语种	中文
文献类型	学位论文
条目标识符	http://210.75.249.4/handle/363003/3108
专题	中国科学院西北高原生物研究所
推荐引用方式 GB/T 7714	李红阁. 高原鼠兔HIF-1α和VEGFs的表达特征及其在低氧适应中的作用[D]. 西北高原生物研究所. 中国科学院西北高原生物研究所,2009.

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