【作者】 赵秀欣；

【导师】 高钰琪；

【作者基本信息】 第三军医大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 世居高原的藏族人群居住高原年代最长且较为封闭,他们具有更完善的氧运输和氧利用的能力,胎儿在宫内发育迟缓少、婴儿出生体重较重、新生儿氧合效率高,是目前公认对于高原低氧环境适应(adaptation)最好的民族[1-2]。有关世居藏族高原低氧适应的机制目前还不清楚。以往的研究显示,高原世居藏族与移居汉族相比,做同等量的功时藏族的耗氧量显著低于汉族,提示藏族在组织、细胞水平对氧的利用率更高,可能是其高原适应的重要机制[3]。我们的研究还发现,线粒体结构和功能的改变是动物习服(acclimatization)低氧环境的重要机制[4]。青藏高原世居藏族在高原低氧环境下已经生活了至少25 000年[3],且由于历史上青藏高原地理环境的封闭性和藏族婚俗的对外隔离性,其遗传学特性得以很好地保存下来。我们推测,世居高原藏族线粒体对于低氧环境的适应性改变可能通过自然选择的作用固定下来,并由此获得了较其他高原世居人群和移居人群更好的对高原低氧环境的适应能力。不过,对于线粒体的研究多在实验动物上进行,关于高原世居者线粒体在低氧适应机制中的作用迄今国内、外均未见文献报道。为此,我们首次在西藏高原地区研究和比较了高原世居藏族和移居汉族胎盘线粒体能量代谢功能,为探索世居高原藏族低氧适应的本质提供新的线索和思路。方法纳入标准月经周期规律、末次月经明确、无烟酒嗜好、孕期37~42周,无高血压、糖尿病、心肺疾病、遗传病及传染病的世居藏族和移居汉族产妇。自愿者分为高原世居藏族组(T组)和高原移居汉族组(H组),两组产妇年龄、体重指数相匹配。比较二组产妇胎盘体积,胎盘重量,胎盘系数,比较二组新生儿低体重出生率,新生儿出生体重和身长。分离提取胎盘线粒体,利用透射电子显微镜观察二组胎盘组织线粒体的超微结构,以Clark氧电极法测定线粒体氧化呼吸活性、复合体I,II,IV活性,以定磷法测定F0-F1 ATP酶活性,高效液相法(HPLC)分析胎盘组织及胎盘线粒体内腺苷酸含量,罗丹明123荧光流动透析法测线粒体膜电位,反转录PCR检测ANTmRNA表达量,Western blot测定ANT蛋白表达,3H-ADP摄入法测定ANT的转运速率。结果1.世居藏族胎盘体积,重量和胎盘系数显著高于移居汉族,世居藏族新生儿出生体重和身长显著高于移居汉族,世居藏族新生儿低体重出生率显著低于移居汉族。2.胎盘线粒体电镜观察:高原藏族线粒体大部分完整,偶尔有线粒体肿胀,空泡移居汉族线粒体广泛肿胀,空泡化,嵴脱落有些可见少量细胞核成分,大多细胞胞浆疏松。3.世居藏族胎盘光镜下表现:绒毛直径较小,表面积增加;绒毛间含丰富的毛细血管,呈血窦样扩张;合体结节形成。移居汉族胎盘光镜下表现:绒毛间质水肿;绒毛间质血管减少;绒毛纤维素增多。4.世居藏族胎盘线粒体复合体I,II及F0F1- ATP酶活性显著高于移居汉族。5.世居藏族胎盘线粒体三态呼吸ST3,呼吸控制率RCR,氧化磷酸化效率OPR显著高于移居汉族。6.世居藏族胎盘线粒体膜电位,胎盘线粒体ATP含量,胎盘组织腺苷酸池ATP/ADP和能荷都高于移居汉族。7.移居汉族ANTmRNA表达显著高于世居藏族。二组ANT的蛋白表达没有差异,世居藏族ANT转运活性显著高于移居汉族。结论1.高原环境影响移居汉族胎儿宫内的生长发育。因此,有必要进一步探索高原环境对移居汉族胎儿生长发育的影响机制,以利于加强移居者围产期保健,降低高原缺氧环境对移居者胎儿生长发育的不良影响。2.在高原缺氧环境中,与世居藏族相比,移居汉族胎盘线粒体呼吸链复合体I,II的酶活性和3态呼吸氧耗速率显著降低,膜电位降低,从而导致氧化磷酸化效率降低,线粒体ATP的合成减少。3.与世居藏族相比,移居汉族胎盘在缺氧环境中,基础氧耗没有增加,ANT核反应增强,这可能是移居汉族高原习服的机制。4.世居藏族胎盘线粒体能量的生成高于移居汉族,这可能是世居藏族对慢性低氧适应的重要机制。更多还原

【Abstract】 The Zang nationality live longest and closed in high altitude.They have more consummate ability of transporting and using oxygen.There are a few intrauterine growth retardation(IUGR).The higer birth weight and the efficient oxygenation make them generally accepted as the best nation of adaptation to high altitude.It is not clear about the mechanism of Zang nationality adaptation to high altitude.It was evidenced that the consumed oxygen by Zang were lower than by Han when they do the coordination duty.This state the better availability in cellular level of Zang may be the important mechanism of adaptation to high altitude.The mitochondrial changes of structure and function is the important mechanism of animal acclimatization to hypoxia environment.The Zang nationality live in high altitude more than about 25 000years,the genetic characteristic of them can be conserved well because of the closed environment and the isolated marriage.We presume the adaptation to hypoxia of Zang nationality maybe fixed through the natural selection.Hence they have the better ability to adaptation hypoxia environment than other nations.The most reseach about mitochondria were done in animal model before.There is no report about the mechanism of mitochondrial adaptation in hypoxia in permanent high altitude native . To expore the mechanism of Zang nationality adaptation to hypoxia,we reseach and compare the placental mitochondrial energy metabolism between nationality Zang and immigrant Han in high altitude.MethodsThe healthy Zang nationality(Z group) and the immigrant Han(H group) pregnant woman were recruited.Their gestation were 37~42 weeks.There is no different of ages and body mass index between them.Comprasion the placental size, the placental weight, the placental ratio, the birth weight, the fetus body lenth, and the low birth rate between the two group.Isolation and abstraction the placental mitochondria, measurement the ST3, ST4, respiratory control rate(RCR), the complexI, II, IV of mitochondrial breath train by Clark oxygen electrode.F0-F1 ATP enzyme activity by measure P, the content of adenine nucleotide in placenta and mitochondria by HPLC; the ANT transport activity by H3-ADP intake; the mitochondrial membrane potential by Rhodamine 123 fluorescence; ANT mRNA expression by reverse PCR; ANT protein expression by Western blot. Observation the placental mitochondrial ultramicrostructure and morphology of the two groups by electro- microscope and light microscope..Results1. The placenta size, weight and placental ratio of Zang nationality are higher than immigrant Han; The birth weiht, the fetus body lenth of Zang nationality are higher than immigrant Han; The low birth weight of Zang nationality are lower than immigrant Han.2. The result of placental mitochondrial electrodemicroscope were: Most of placental mitochondria structure of Zang nationality are integraty, while most of placental mitochondria structure of immigrant Han are swelling, vacuolus, cristae shedding, most kytoplasm raritas.3. The result of placental mitochondrial morphology of Zang nationality were: the smaller diameter of villus, the bigger surface area of villus, the affluent capillary between the villus, zoarium tuberculation. The result of placental mitochondrial morphology of immigrant Han were: the villus interstitial edema, the less capillary of villus interstitial, the increased villus fiber.3. The activity of placental breath train complex I, II, and F0-F1 ATPase enzymatic of Zang nationality are higher than that of immigrant Han.4. The ST3, RCR, OPR of are higher than that of immigrant Han.5. The placental mitochondrial membrane potential, the content of ATP in placenta and mitochondria of Zang nationality are higher than those of immigrant Han; the placenta adenylate pool ATP/ADP and energy charge of Zang nationality are higher than those of immigrant Han.6. The ANT mRNA expression of Zang nationality are lower significantly than that of immigrant Han; the ANT protein expression is no difference between the two groups; ANT transport activity of Zang nationality are higher than that of immigrant Han.Conclusion1. The fetus development of immigrant Han are effected by high altitude environment.In order to enhance the perinatal care of the immigrant and reduce the harmful effecion of the hypoxia in high altitude, so it’s nessasery to explore the mechanism of this effection.2.Compared with the Zang nationality, under the hypoxia environment, the placenta mitochondrial ST3, RCR, membrane potential of immigrant Han was decreased, lead to reduce the ATP synthesis.3.Compared with Zang nationality, the basic oxygen consumption didn’t increase and the expression of ANT mRNA increased in the immigrant Han , which maybe be the acclimatization mechanism to high altitude of immigrant Han.4.It’s maybe the important mechanism of adaption to hypoxia in high altitude of Zang nationality that the placental mitochondrial energy metabolismo of them are higer than that of immigrant Han.更多还原