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线粒体基因组与高原习服适应相关性的初步研究

The Initial Research about the Relationship between Mitochondrial Genome and High Altitude Acclimatization and Adaptation

【作者】 罗勇军

【导师】 高钰琪;

【作者基本信息】 第三军医大学 , 病理生理学, 2008, 博士

【摘要】 高原环境影响机体的主要因素是缺氧,机体对高原环境的习服适应也主要是围绕着氧的摄取-运输-利用这条轴线来进行的。线粒体是机体能量代谢的中心,是组织、细胞氧利用的关键场所,机体耗能的90%以上来自于线粒体的氧化磷酸化作用。因此,线粒体作为细胞的“动力工厂”,在低氧引起细胞损伤和组织、细胞对低氧环境的习服过程中的作用都是至关重要的。以往的研究发现急性缺氧可以抑制线粒体DNA(mitochondrial DNA,mtDNA)的转录和翻译,损害线粒体的功能,而慢性缺氧时线粒体的功能得到一定程度的恢复,这种线粒体的功能改变是机体低氧习服适应的一个重要机制,线粒体的功能改变受到线粒体基因的表达变化、线粒体基因组序列和数目变化的影响。高原世居藏族是目前公认的对高原低氧环境适应最好的民族,有关高原世居藏族低氧适应的机制目前还不清楚。高原世居藏族已建立了完善的母体——胎盘——胎儿系统及适应低氧的胎盘机制,因而采用基因芯片技术对高原世居藏族胎盘组织线粒体功能相关基因的表达谱变化进行深入研究,有助于我们探讨线粒体基因组在高原适应中的机制。线粒体功能相关基因表达谱发生了变化可能是由于线粒体基因组变化所致。高原鼠兔(ochotona curzoniae)是目前研究高原习服适应的理想动物模型之一,高原鼠兔在长期的低氧和寒冷环境中可能通过提高线粒体基因组的进化速度和改变OXPHOS的效率获得高原适应,对于高原鼠兔的mtDNA的全长序列缺少报道,对高原鼠兔线粒体基因组与高原适应机制尚无研究。因而采用PCR-测序的方法测定高原鼠兔的线粒体基因组及研究该基因组与高原适应的关系。线粒体是半自主细胞器,其功能由线粒体基因组和核基因组共同决定。与核DNA(nuclear DNA,nDNA)相比,mtDNA突变率较高,在这些突变中有的突变为中性突变,这种中性突变逐渐累积就形成单倍群(haplogroup)和单倍型(haplotype)。一些线粒体的单倍群或单倍型能够影响线粒体的功能,例如使ATP和ROS的生成发生改变,因而研究人线粒体单倍群、单倍型与高原习服适应以及高原习服不良的疾病(高原肺水肿)的关系,为深入研究高原习服适应的线粒体遗传机制打下基础。本研究中首先通过对26例汉族个体的mtDNA全长进行测序,序列进行比对分析,寻找出汉族人mtDNA多态性的特征(SNPs或单倍群);然后采用多聚酶链式反应-限制性内切酶多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)、多聚酶链式反应-高温连接酶反应(polymerase chain reaction-ligase detection reaction,PCR-LDR)的方法分别研究汉族人线粒体的单倍群和单倍型与高原适应以及高原习服不良的疾病(高原肺水肿)的易感性之间的关系。线粒体基因组的变化处序列变化外还有数目变化,线粒体呼吸链氧化磷酸化不但与mtDNA结构的完整性相关还与其拷贝数相关,mtDNA拷贝数增加被认为是总线粒体呼吸功能的代偿。对于mtDNA拷贝数在高原习服适应中变化规律,目前尚不清楚。采用定量PCR的方法研究线粒体基因组数目多态性与高原习服适应的关系。我们取得的主要结果有:1.高原世居藏族与移居汉族相比,有24个基因的表达存在差异,其中表达上调的基因3个,表达下调的基因21个。这些差异表达基因编码蛋白的功能涉及能量代谢、信号转导、细胞增殖、电子链传递、DNA损伤与修复、细胞粘附分子等方面。通过qRT-PCR发现Kdr、Dctn2以及Cox17基因的mRNA表达与基因芯片结果同趋势。2.高原鼠兔mtDNA的全长为17,131 bp(No.EF535828),与其它哺乳动物的mtDNA序列相似,含13种编码蛋白质、22个tRNA和2个rRNA。除ND6和8个tRNA基因(Gln-tRNA、Ser-tRNA、Ala-tRNA、sn-tRNA、Cys-tRNA、Tyr-tRNA、Glu-tRNA以及Pro-tRNA)位于轻链上,其余基因位于重链上。该序列碱基A占31.02%,碱基T占29.65%,碱基C占13.46%,碱基G占25.87%。进化分析发现高原鼠兔与美洲鼠兔的遗传距离最近,其次是与家兔的遗传距离较近;通过对高原鼠兔的氨基酸序列分析发现,高原鼠兔的COX1、COX2基因的氨基酸序列发生了改变。3.高原世居藏族中单倍群D4的频率(4.5%)非常显著低于平原汉族(20.0%,P<0.005)和移居汉族(16.4%,P<0.05),单倍型nt3010G-nt3970C在高原世居藏族中的频率(84.8%)非常显著高于平原汉族(51.4%,P<0.005)和移居汉族(63.3%, P<0.005)。单倍群D4和单倍型nt3010G-nt3970C的频率在平原汉族和移居汉族中没有差别。4. HAPE病例组单倍群D4的频率(14.2%)显著低于对照组(22.9% , P=0.033, OR=0.555, 95%CI: 0.327-0.942);HAPE病例组单倍群B的频率(21.6%)显著高于对照组(11.5%,P=0.013, OR=2.118, 95%CI: 1.194-3.756);在单倍群R9中HAPE病例组mtDNA等位基因nt13497G频率(19.4%)高于对照组(0%,P<0.05);HAPE病例组单倍型nt3970C-nt13497G的频率(15.6%)非常显著高于对照组(0%,P<0.005)。5.缺氧SD大鼠(4500 m,30天)肝脏mtDNA拷贝数(2081.86±435.6060)非常显著高于高原鼠兔(51.72±13.6977,P<0.005)和平原对照SD大鼠(49.13±17.0393,P<0.005)。6.在高原习服过程中mtDNA拷贝数增加,对于获得高原适应高原世居藏族人,mtDNA拷贝数又逐渐降低。HAPE病人mtDNA拷贝数低可能是HAPE的发病原因和遗传标记。7.移居汉族的mtDNA拷贝数(0.1238±0.0180)显著高于平原汉族(0.0822±0.0094,P<0.05) ; HAPE病人的mtDNA拷贝数(0.0291±0.0055)非常显著低于平原汉族(0.0822±0.0094 , P<0.005)、移居汉族(0.1238±0.0180 , P<0.005)和高原世居藏族(0.0847±0.0128,P<0.005)。通过上述研究我们可以得到以下结论:1.线粒体基因的表达变化可能改变线粒体COXI-COXIV的活性而影响ATP的生成,参与高原适应。2.高原鼠兔mtDNA序列的改变可能是其高原适应的重要机制。3.首次报道了汉族人mtDNA 9个SNPs位点。4.单倍群D4与世居藏族高原适应呈负相关,单倍型nt3010G-nt3970C与世居藏族高原适应呈正相关。5.单倍群D4为HAPE的保护因素,单倍群B和单倍型nt3970C-nt13497G为HAPE的危险因素。6.在高原习服过程中mtDNA拷贝数增加,获得高原适应后mtDNA的拷贝数又降低。7. HAPE病人mtDNA拷贝数低可能是其发病的重要遗传机制。总之,本文从线粒体基因组序列和数量变异等多方面探讨线粒体基因组与高原适应的相关性,推测线粒体基因组序列和数量变化均会影响线粒体功能,这为低氧习服适应机制研究和寻找促低氧习服措施提供分子标志和靶基因。

【Abstract】 Oxygen utilization is one of the central links of hypoxia adaptation in high altitude. Mitochondria house is the final biochemical steps in the production of reducing equivalents that react at the terminal oxidizes of the respiratory chain with molecular oxygen, and thus have been proved to be a reactive organelle in hypoxia. Morphological and functional changes of mitochondria and impairment of mitochondrial DNA (mtDNA) were also been found in acute hypoxia, which could be partially repaired as hypoxia prolonged. Indeed, ultrastructural data obtained from rat and human tissues exposed to hypobaric hypoxia revealed significant mitochondrial morphological changes, namely considerable swelling and cristae degeneration. The mitochondrial function changes are the most important mechanism for high altitude acclimatization and adaptation. Whether the mitochondrial function changes related to the mitochondrial related genes expression changes, mtDNA sequence and copy number variation need us to study.The native Tibetan had lived the longest on the Tibetan plateau, with more consummate ability of transporting and using oxygen, hence they have the better ability to adaptation hypoxia environment than other nations. There was little report about the mitochondrial mechanism of hypoxia adaptation in the high altitude native Tibetan fetuses, to our knowledge. The placentas of native Tibetan and the high-altitude Han (ha-Han) were collected, after the total RNA extraction; the finally synthesized cDNAs were hybridized to mitochondrial array to find the different expression genes between them. Then, the Cox17,DCTN2 and KDR were chosen at random from the different expression genes to further verify the array results using the SYBR Green real-time PCR.Pikas originated in Asia and are small lagomorphs native to cold climates. The plateau pika, Ochotona curzoniae is a keystone species on the Qinghai-Tibet Plateau and an ideal animal model for hypoxic adaptation studies. Altered mitochondrial function, especially cytochrome c oxidase activity, is an important factor in modulation of energy generation and expenditure during cold and hypoxia adaptation. In this study, we determined the complete nucleotide sequence of the O. curzoniae mitochondrial genome.Tibetans are considered the best adapted to the high altitude environment. Mitochondrion is one of the central links of oxygen consumption and mtDNA variation may play a role in high altitude adaptation. In addition, alleles at several polymorphic sites in mtDNA define some common haplogroups, and some of these haplogroups have been implicated in the risk of developing several diseases. The relationship between the mitochondrial haplogroup/hapotype and high altitude adaptation in Tibetan or HAPE susceptibility were studied in this study.Then the relationship between the mtDNA copy number variation and high altitude acclimatization / adaptation in the animals or human was studied in this studied.The main results of our study were listed as follow:1. By a standard of≥1.5 or≤0.67, there were 24 different expressed genes between the native Tibetan and the ha-Han placentas, including 3 up-regulated genes and 21 down-regulated genes. These genes were related to energy metabolism, signal transduction, cell proliferation, electron transport, cell adhesion, nucleotide-excision repair. The array results of Cox17, DCTN2 and KDR were further verified by the real-time RT-PCR.2. The plateau pika mitochondrial DNA is 17,131 bp long and encodes the complete set of 37 proteins typical for vertebrates. Phylogenetic analysis based on concatenated heavy-strand encoded protein-coding genes revealed that pikas are closer to rabbit and hare than to rat.3. The mitochondrial haplogroup D4 frequency was low in Tibetan (P=0.001 VS la-Han, OR=0.166, 95% CI=0.048-0.567; P=0.009 VS ha-Han OR=0.232, 95% CI=0.069-0.778). The characteristic haplotype nt3010G-nt3970C was the significantly higher in Tibetan than the La-Han (P=0.000) and Ha-Han (P=0.001).4. The frequency of haplogroup D4 was lower in the HAPE patients (P=0.033, OR=0.555, 95%CI: 0.327-0.942) than in the controls, however the haplogroup B was higher in the HAPE patients (P=0.013, OR=2.118, 95%CI: 1.194-3.756) than in the controls. The allele nt13497G was significantly higher in the HAPEs (P=0.012) than in the controls among haplogroup R9. The haplotype nt3970C-nt13497G was the higher in the HAPE patients (P=0.000) as compared with the controls also.5. The mtDNA copy numbers in the control SD rats were lower than plateau pika (P<0.005) and SD rat (4500 m, 30 d) (P<0.005).6. The mtDNA copy numbers in the la-Han were lower than ha-Hans (P<0.05). Moreover, the mtDNA copy numbers in the HAPE patients were lower than that of la-Hans (P<0.005), ha-Han (P<0.005) and native Tibetan (P<0.005).The conclusion as suggested in our study was followed:1. The altered mitochondrial related genes in the native Tibetan placentas may have a role in the high altitude adaptation through changing the activity of COX.2. The rabbit or hare would be a good control animal for pikas in cold and hypoxia adaptation studies. Fifteen novel mitochondrial DNA-encoded amino acid changes were identified in the pikas, including three in the subunits of cytochrome c oxidase.3. The mtDNA nucleotides sites (2706, 7028, 8860, 11719, 15326) were totally different from rCRS, 48 SNPs were with the frequency over 5% from the 26 whole mtDNA sequences analysis. These findings provided new insights into the characteristics of Han Chinese mitochondrial genetic diversity.4. The mitochondrial haplogroup D4 is negative associated with high altitude adaptation in Tibetan; however haplotype nt3010G-nt3970C was the significantly good to high altitude adaptation in Tibetan. Our findings suggest that Tibetans process unique mitochondrial variations which may be genetic background associated with high altitude adaptation.5. The haplogroup D4 was the protection factor to HAPE susceptibility, however haplogroup B, allele nt13497G and haplotype nt3970C-nt13497G were the risk factors to HAPE susceptibility, which could contribute to defining the role of the mitochondrial genome in HAPE pathogenesis. 6. The mtDNA copy number increased during the hypoxia acclimatization, but it decreased in the samples that had hypoxia adaptation. In the HAPE patients, the mtDNA copy number was low, which could contribute to define the role of the mitochondrial genome in HAPE pathogenesis.

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