“大通牦牛”胎盘与胎儿心肌线粒体低氧耐受机制的比较发育学研究

Da Tong yak is the first new national yak breed that was bred successfully utilizing genetic resource of the endemic wild yak of Qinghai Province，which has stable herediry and strong stress-resistance characteristic. In the early development of animal, the fetus is easy to be affected by environmental factors, and will take predictive adaptive responses to adapt itself, and the structures and functions change of mitochondrial is the critical mechanism that animal sacclimatizes hypoxia environment. In this project, placenta and fetal myocardial tissue(second trimester and 1 day birth) of Datong yak will be selected as research object compared with domesticated yak at the same altitudinal gradients. The transmission electron microscope will be used to observe mitochondrial stereoscopic morphological structures of placenta and fetal myocardial tissue. The histology and immunohistochemistry methods will be adopted to analyze their histomorphological structure, microvascular density and vascular endothelial growth factor. The fluorescent quantitative PCR will be applied to detect the expressions pattern of cytochrome oxidase（COX）and succinate dehydrogenase(SDH) subunits which is the maker enzymes of mitochondrial inner membrane. Simultaneously, protein expression level of hypoxia inducible factor-1 will be analyzed, which is the key regulatory effect under the condition of hypoxic adaptation. The aim of this project is to clarify the biological mechanism of placental-fetal oxygen supplying system in adaptation to the hypoxia environment during the early development period of Datong yak from cell-molecular-whole level, and to provide theoretical data to protect plateau endemic animal species resources.

"大通牦牛"是利用我省特有野牦牛资源培育成功的第一个国家级牦牛新品种，具有稳定的遗传性。动物在胎儿发育早期可受到环境因素的影响，胎儿会采取"预测适应反应"，而线粒体结构和功能的改变是动物习服低氧环境的重要机制。本项目拟选（妊娠中期和出生1日龄）大通牦牛胎盘及胎儿心肌组织为研究对象，并与同海拔梯度家养牦牛进行比较。应用透射电镜观察胎盘及胎儿心肌线粒体立体形态学结构；应用组织学和免疫组化方法分析其组织形态学特点、微血管密度（MVD）及血管内皮生长因子(VEGF)；利用Real-time PCR和Western blotting方法探讨线粒体内膜酶细胞色素C氧化酶（COX）和琥珀酸脱氢酶（SDH）亚基的表达调控及组织中低氧诱导因子（HIF-1α）蛋白表达模式。目的在于从细胞-分子-整体水平探讨大通牦牛发育早期"胎盘-胎儿"供氧系统对低氧环境生态适应的生物学机制，为保护高原特有种质资源提供理论支撑

细胞呼吸对低氧的适应表现在呼吸链酶学特征和线粒体的结构功能的变化上，而琥珀酸脱氢酶（SDH）和细胞色素C氧化酶（COX）是权衡线粒体特性及功能的标志性酶。为揭示“大通牦牛”发育早期“胎盘-胎儿”线粒体供氧系统对青藏高原极端环境压力下生态适应的生物学机制，本项目选取大通牦牛（海拔3200m）妊娠4月龄胎儿和出生1日龄犊牛心肌组织和胎盘组织作为研究对象，并与环湖型家牦牛（海拔3200m）和高原型家牦牛（海拔3700m）进行比较，运用组织学技术对胎盘及胎儿心肌线粒体的形态学特点进行比较，运用分子生物学技术探讨线粒体细胞色素C氧化酶（COX）和琥珀酸脱氢酶（SDH）亚基的表达调控及组织中低氧诱导因子（HIF-1a）蛋白表达。研究结果显示：（1）妊娠4月龄胎儿心肌线粒体与1日龄犊牛相比，表现为体积小、数量多的特点；（2）1日龄犊牛心肌中COX-1、COX-II基因表达量均高于4月龄胎儿；（3）出生后，心肌组织中的COX-I、II mRNA表达量随年龄的增加呈上调趋势；而出生1日龄时COX-I、II mRNA表达量最高；（4）出生后，心肌组织中SDH 4个亚基转录水平随年龄的增长均表现为上调趋势；而出生1日龄犊牛SDH 4个亚基表达量均高于其他年龄段；组织线粒体SDH 4个亚基mRNA表达量由高到低依次为SDHA、SDHB、SDHC和SDHD；（5）出生后，HIF-1α mRNA表达量随年龄增长表达量稳定增加；出生1日龄和6月龄牦牛HIF-1α mRNA表达量显著高于其他年龄组；（6）不同海拔梯度妊娠4月龄胎儿各组织HIF-1α mRNA和蛋白的表达量随海拔升高而上调。而出生后，1日龄大通牦牛各组织HIF-1α mRNA和蛋白的表达量最高；（7）妊娠4月龄胎儿和1日龄犊牛的COX1、COX2、COX3三个基因在心肌组织的表达量最高，脾脏和肺脏的表达量最低。