牦牛高原低氧适应因子对其红细胞低氧适应性的分子调控机制

Yak is a kind of typical indigenous animal with good adaptability to high altitude and low oxygen environment in Qinghai-Tebetan Plateau. The molecule mechanism of hypoxia adaptation is unclear in yak erythrocyte. In order to find out the gender/age-related characteristics in every growth and development stage, the plateau adaptation factors (PHD family, HIF family, HO family and EPO) will be measured in blood and various organs (liver and kidney) of male and female yak by Real-time quantitative PCR, immunohistochemistry, ELISA and Western-blot. The impacts of hypoxic plateau on plateau hypoxia adaptation factors will be evaluated in yak blood and major related organs by comparing the cattle in low altitude and the cattle-yak (yak and cattle hybrids) in higher altitude. To figure out the plateau hypoxia adaptation factors pathway in normoxia and hypoxia conditions, HIF family level will be detected in yak primary hepatocytes and renal tubules with decreased PHD (PHD2 and PHD3) by RNAi and increased by gene overexpression techniques; HO (HIF-1α and HIF-2α) and EPO will also be test with HIF down-regulation and up-regulation. This program will supply basic theory for the further study on plateau hypoxia adaptation.

牦牛是一种充分适应高海拔低氧环境的青藏高原典型土著动物，其红细胞的低氧适应分子调控机制值得探究。本研究拟首先采用实时荧光定量PCR、免疫组织化学法、ELISA和Western-blot技术，检测不同性别和年龄的牦牛生长发育过程中血液及主要相关器官中高原低氧适应因子表达模式的差异，并通过比较生活在不同海拔高度的黄牛及黄牛与牦牛的杂交后代犏牛的高原低氧适应因子表达的差异，揭示调控红细胞的高原低氧适应因子之间的关系。在此基础上，采用RNA干扰和基因过表达技术下调或上调牦牛体外培养肝细胞及肾小管周围成纤维细胞中高原低氧适应因子的表达，通过实时荧光定量PCR和Western-blot技术检测含氧正常及低氧条件下高原低氧适应因子的表达情况，揭示牦牛高原低氧适应性因子对其红细胞的调控通路。本研究将为揭示高原哺乳动物红细胞的低氧适应性分子调控机制奠定理论基础。

红细胞的低氧适应性分子调控是牦牛适应高海拔低氧环境的重要机制之一。采用使用实时荧光定量PCR、免疫组织化学法、Western blot、RNA干扰、转录组学和蛋白质组学分析等技术，对牦牛低氧适应性因子的表达特征、EPO的表达调控、EPO生成器官的结构特征和HIF-1α、HO-1在牦牛肺脏低氧适应性中的发挥的作用及调控机制进行了系统的研究。（1）阐明了促红细胞生成素（EPO）、低氧诱导因子-1α和2α（HIF-1α和HIF-2α）、血红素加氧酶（HO-1）和脯氨酰羟化酶（PHD）是牦牛低氧适应性调控的关键因子，在成年牦牛各主要器官中均有所表达，但各因子表达量及表达位点有所不同。（2）揭示了肾间质成纤维细胞是牦牛肾脏中EPO生成细胞，EPO的表达不仅受PHD—HIF-2α轴的调控，亦受HSP90的调控；HIF-1α表达的增高对牦牛肾小管上皮细胞上皮-间质转化具有促进作用。（3）牦牛肾脏动脉系统和集合系统均有其自身特点。与黄牛相比，牦牛动脉管壁发生平滑肌相对增生变厚和动脉管壁特异性重塑的现象，可能是其维持血管壁发挥正常生理功能的适应性变化。（4）发现HIF-1α和血红素加氧酶HO-1在牦牛肺脏中的表达量呈增龄性升高，并且主要定位于肺动脉平滑肌细胞（PASMCs）中；低氧不仅可以促进PASMCs的增殖和抗凋亡，还可诱导HIF-1α的瞬时表达。HIF-1α和HO-1均参与了低氧诱导牦牛PASMCs的增殖和抗凋亡过程，HO-1可抑制低氧诱导的牦牛PASMCs增殖，而HIF-1α则发挥了相拮抗的作用。（5）低氧处理牦牛PASMCs的转录组文库，首次系统阐明了HIF-1α信号通路、TGF-β信号通路和糖酵解/糖异生等通路参与了PASMCs的低氧适应过程。（6）利用蛋白质组学的TMT技术分析了常氧组和低氧组的牦牛PASMCs，首次发现DEPs主要与DNA复制、细胞周期和TGF-β / HIF-1α / MAPK / NF-κB等信号通路有关；