HIF-1α和eNOS基因多态性与个体低氧训练效果的关联性研究

目的：拟从基因多态性角度探索人类个体低氧训练效果差异性的分子生物学机制。方法：选取41名健康受试者在模拟海拔2500m高度进行4周“高住-高练-低训”（低氧暴露10h／d,3次70％VO2max低氧训练／周,共4周）,观察低氧诱导因子-1α基因（HIF-1α）C1772T多态性、内皮型一氧化氮合酶基因（eNOS）第4内含子27bp（4b／a）可变数目重复性多态性（VNTR）与低氧训练期间最大摄氧量（VO2max）、血红蛋白（Hb）、红细胞数（RBC）及血氧饱和度（SpaO2）等生理指标变化的关联性。结果：携带CT基因型和CT＋ba复合基因型的受试者,4周低氧训练后△VO2max显著增加（P〈0．05）,CT基因型受试者低氧训练前、后△RBC,△Hb无显著性差异,但较其它基因型相比,表现出升高趋势;CT基因型与ba基因型受试者在低氧定量负荷实验中,SpaO2表现出高于其它基因型的趋势,但无显著性差异。结论：HIF-1α基因C1772T多态性及eNOS基因4b／a多态性可能与低氧训练效果及低氧环境下适应能力的个体差异性有关,CT基因型及CF5ba复合基因在低氧适应能力上具有一定的优势。     

高原低氧对机体无氧代谢阈值的影响

为了探讨高原低氧对机体无氧代谢阈值(AT)的影响,本研究采用Wasserman无创性方法,分别测定了11名新兵在平原(四川淮口,海拔500m)和经空运进驻高原 (西藏错那,海拔4370m)后的第3、5、7和14天的AT。结果表明:新兵进驻高原后AT由平原的813.6±147.4kg·m/min降低到395.5±194.5 kg·m/min(P<0.01);高原低氧引起AT的降低幅度与受试者平原AT的高低呈正相关(r=0.933,P<0.01);进驻高原后第3、5、7天AT维持在较低水平,随后呈上升趋势。但移居高原1年战士的AT仍低于平原水平(P<0.05)。提示,高原低氧能够显著地降低机体的AT,并且AT越高的个体进驻高原后受低氧环境的影响程度越大。     

高原训练中运动员腹泻发生状况及肠道菌群的变化

目的:通过实地调研考察高原训练中运动员腹泻发生率,并采集运动员粪便样品,研究高原训练过程中运动员肠道菌群变化。方法:赴多巴国家高原训练基地调研在训运动员胃肠道反应,统计腹泻发生率及发生的时间特点。分别在运动员进驻高原前和高原训练过程中采集粪便样品,进行粪便常规检测、提取全基因组DNA,质检合格样品采用高通量测序的手段进行微生物多样性分析。结果:1)高原训练期间运动员腹泻发生率为46.7%,所有腹泻案例持续时间均10天,其中74.1%持续时间5天。2)腹泻案例发生主要集中在两个时间段,分别是进入高原后的第1-3天(占总腹泻案例数的35.7%)及进入高原后的第7-10天(占总腹泻案例数的46.4%),至进入高原后的第12天不再有新增腹泻案例出现。3)运动员进驻高原前后微生物多样性无显著差异。4)相较与平原时,运动员肠道菌群拟杆菌门先上升后恢复,厚壁菌门先下降后恢复。结论:1)高原训练中运动员多发腹泻,腹泻案例呈现出两个爆发高峰。2)运动员进驻高原后肠道菌群结构发生改变,表现为拟杆菌门占比上升、厚壁菌门占比下降。随着微生物多样性分析技术的普及,针对性补充有益菌保障运动员平稳度过高原习服期,有助于降低高原训练期间腹泻发生率。     

低氧低二氧化碳对大鼠脑血流的调节作用

低氧下脑血流与急性高原病有关,许多研究已证明,低氧不适应者的ＣＢＦ明显高于低氧适应者,而高原世居者则低于海平正常值。低氧下低动脉血二氧化碳分压有对抗低氧扩血管及降低ＣＢＦ的作用。因此研究低氧下低ＰａＣＯ２对ＣＢＦ的调节作用对于研究急性高原病的防治及其机理分析具有重要意义。本项研究目的：（１）揭示低氧下低ＰａＣＯ２与ＣＢＦ之间的关系;（２）通过增加通气量使ＰａＣＯ２下降至合适水平,以保护脑血流维持在     

高原低氧条件下不同时间大鼠海马CA1区神经细胞粘附分子的表达

目的:观察高海拔低氧条件下不同时间大鼠海马CA1区神经细胞粘附分子的表达变化,探讨NCAM在机体对低氧应激反应中的作用。方法:将平原SD大鼠运至海拔(4100m)地区,在第2、5、9、15天取大鼠海马,常规免疫组化及RT-PCR检测高原环境下NCAM的表达变化。结果:NCAM在高海拔大鼠海马CA1区神经细胞NCAM的表达在第2、5、9天是明显低于正常(P0.05),在第15天达到正常(P0.05)。结论:高原低氧应激反应后NCAM基因表达先降低后升高,提示其在神经损伤修复过程中可能起重要作用。     

西红花苷对急性高海拔低氧条件大鼠脑海马PGC-1α表达及凋亡的影响

本研究探讨西红花苷提前干预用药对急性高海拔低氧条件下大鼠脑海马组织的影响。以西红花苷药效机制为研究内容,通过组织病理及透射电镜超微结构形态学观察,RT-PCR和Western blotting检测PGC-1α、TFAM、Bcl-2、Bax、Caspase-3在mRNA和蛋白水平表达,TUNEL凋亡检测海马神经元凋亡水平。研究发现,在低氧模型组海马组织在第1天、第3天、第5天、第7天细胞结构有不同程度紊乱,线粒体损伤,以第1天、第3天最为显著,而西红花苷组线粒体损伤明显改善;PGC-1α、TFAM、Bcl-2相对表达量比较发现西红花苷组在mRNA和蛋白水平表达在第1天、第3天和第5天明显高于低氧模型组(p0.05),而第7天两组相对表达量差别无统计学意义(p0.05);比较Bax、Caspase-3在第1天、第3天和第5天m RNA和蛋白水平相对表达量发现西红花组相明显低于低氧模型组(p0.05),而第7天两组相对表达量差别无统计学意义(p0.05);TUNEL检测发现脑海马CA1区神经元凋亡率与低氧模型比较西红花苷组在第1天、第3天、第5天比较均较低(p0.05),表明西红花苷通过抗线粒体损伤及凋亡具有保护急性高海拔低氧大鼠脑海马的药理作用,为研究高原病的防治提供理论依据。     

习服高原与脱习服

目的:研究人体进入低氧、低气压、低温的高海拔环境后机体出现生理应激反应和习服高原的过程。并探索在高原居住若干年后返回低海拔常氧地区机体呈现脱高原习服的临床征象。方法:习服高原环境采用回顾在藏工作50年资料、摘取相关数据,青藏铁路施工中调查资料、空运部队进藏公开发表资料之汇总共5万例。脱高原习服采用曾在藏工作生活过3~50年返回平原后问卷调查2 060人(分布全国各地)的综合资料。结果:①习服高原环境方面:5万例资料显示:当人进入高原低氧环境出现生理性的调节,临床上出现轻微的高原反应。也有个别人根本无任何反应。若体内缺氧状况得不到及时改善,原属生理应激反应就转变为病理生理范畴,出现重症高原反应,进一步发展可发生急性高原肺水肿、高原脑水肿,应迅速送医院治疗,贻误会造成死亡。在已习服高原人群中若干年后有10%左右的移居者出现红细胞过度增生,血红蛋白超过210mg/L,肺动脉收缩压大于50mmHg,右心增大,呈"多血貌"的慢性高原病(蒙赫氏病)。脱高原习服方面:我们采用在西藏曾已习服高原3~50年后返回平原2 060人,分布全国各地已完成脱高原习服和正在脱高原习服者问卷调查临床征象。②脱高原习服的症状轻重及持续时间的久暂与在高原环境海拔高度、年龄及习服高原时间久暂成正相关。一般情况下三年后就可完全脱高原习服,也有极少数人1‰无法脱习服,在低原无法生活而重返高原。在脱习服人群中有200人分布在黄土高原,这里平均海拔1 000m以上,这些人反应极轻微,况三个月后就完全脱高原习服。结论:习服高原最佳选择阶梯性适应训练法进入高原。脱习服如能选择阶梯性适应到低海拔地区,则可减少脱习服时间与减轻症状。     

模型鼠低氧预适应适宜氧气浓度研究

目的:研究低氧预适应训练的适宜氧气浓度。方法:设计了短期和长期两种间歇性低氧暴露模式,研究了一系列不同浓度的低氧环境对模型鼠体重、血氧饱和度、游泳能力等方面的影响,进而探讨低氧预适应效应与氧气浓度之间的内在联系。结果:模型鼠长期暴露于低氧环境中,其体重增长率逐步下降;在15%～8%的低氧浓度区间,模型鼠血氧饱和度随氧气浓度降低呈现平台似缓慢下降趋势;低氧预适应训练后的模型鼠游泳能力显著提高,经在10%低氧环境中进行低氧预适应训练后的昆明小鼠游泳能力提高最为明显。结论:适当浓度的低氧预适应训练可以改善模型鼠低氧耐受能力,显著提高模型鼠运动能力。15%～10%氧气浓度区间可视为低氧预适应有益作用区间。10%氧气浓度为模型鼠低氧预适应训练的较适宜浓度。     

低氧反应和屏气反应在急性高原反应预测中的作用

目的:探讨低氧反应和屏气反应在急性高原反应预测中的作用.方法:在平原观察113名入藏人员吸入10%低氧气体10 min和屏气过程中的血氧饱和度、心率和血压变化;进入高原后进行急性高原反应(AMS)症状评分;两组数据进行相关分析,并对急性高原反应者和基本无反应者数据进行分析.结果:吸入10%低氧气体过程中,动脉血氧饱和度进行性下降,心率迅速升高,血压普遍呈现先升高后降低的变化趋势.急性高原反应者吸入低氧气体1min时的心率明显慢于基本无反应者.所观测指标均存在着较大的个体差异,但相关分析表明,AMS评分仅与吸入低氧气体7 min时的心率呈显著负相关,相关系数r为-0.176.结论:在平原单纯用低氧和屏气反应来预测3 658 m高原地区的AMS发病情况意义可能有限.     

低氧暴露诱导肌萎缩发生的机制探讨

高海拔地区常伴随着诸多不利环境因素,使初上高原者处于应激状态,对机体代谢系统产生一系列不良影响,其中之一就是诱发骨骼肌萎缩。肌萎缩是一种肌肉功能减退的反应,表现为肌纤维横截面积减小,肌纤维类型转变和肌肉力量、耐力下降。高原环境造成的肌萎缩与低氧环境密切相关。高原训练是竞技体育中一种提高氧运输能力、心脏供血能力及最大摄氧量的有效训练方式,但此过程中造成的骨骼肌质量丢失会影响运动员力量和耐力的发挥,不利于运动表现;同时,随着高原旅游和低氧减肥的兴起,世居平原大众进入高原后发生的肌量丢失和肌力下降也会影响其健康状态。低氧暴露所诱发的肌萎缩程度由低氧浓度和暴露时长所决定,是一个多器官、多组织参与的整体调控骨骼肌蛋白代谢失衡的过程,且在不同类型的肌纤维中表现不同。但目前关于低氧暴露导致肌萎缩的机制还不完全清楚。因此,本文将就该问题相关研究进展进行综述,以期进一步阐明低氧诱导肌萎缩的生物学机制,从而利用低氧刺激更好地提高运动成绩和服务大众健康。     

A genetic adaptive pattern＊low hemoglobin concentration in the himalayan highlanders

Mean hemoglobin （Hb） concentration of about 3 500 subjects derived from 17 studies of Himalayan highlanders （Tibetans, Sherpas, and Ladakhis） was compared with lowlanders （Chinese Han, Indian Tamils） lived in the Himalayas, and European climbers during Everest expeditions as well as Andean natives. The results found that Hb concentration in Himalayan highlanders was systemically lower than those reported for Andean natives and lowland immigrants. These comparative data demonstrated that a healthy native population may successfully reside at high altitude without a significant elevation in Hb, and the lower Hb levels of Himalayan highlanders than those of migrated lowlanders and Andean natives are an example of favourable adaptation over the generations. In addition, excessive polycythemia has frequently been used as a marker of chronic mountain sickness （CMS）. Altitude populations who have a higher Hb concentration also have a higher incidence of CMS. The low Hb in Himalayans suggested as showing adaptation over many generations in Tibetan stock. Recent work in Tibet, suggested that Tibetans there may have adapted to high altitude as a result of evolutionary pressure selecting for genes which give an advantage at altitude. All of the population genomic and statistical analysis indicated that EPAS1 and EGLN1 are mostly likely responsible for high altitude adaptation and closely related to low Hb concentration in Tibetans. These data supported the hypothesis that Himalayan highlanders have evolved a genetically different erythropoietic response to chronic hypoxia by virtue of their much longer exposure to high altitude.     

Emerging roles of miR-210 and other non-coding RNAs in the hypoxic response

Hypoxia is a key component of the tumor microenviron- merit and represents a well-documented source of thera- peutic failure in clinical oncology. Recent work has provided support for the idea that non-coding RNAs, and in particular, microRNAs, may play important roles in the adaptive response to low oxygen in tumors. Specifically, all published studies agree that the induction of microRNA- 210 （miR-210） is a consistent feature of the hypoxic re- sponse in both normal and malignant cells, miR-210 is a robust target of hypoxia-inducible factors, and its overex- pression has been detected in a variety of diseases with a hypoxic component, including most solid tumors. High levels of miR-210 have been linked to an in vivo hypoxic sig- nature and to adverse prognosis in breast and pancreatic cancer patients. A wide variety of miR-210 targets have been identified, pointing to roles in mitochondrial metabol- ism, angiogenesis, DNA damage response, apoptosis, and cell survival. Such targets are suspected to affect the devel- opment of tumors in multiple ways; therefore, an increased knowledge about miR-210＇s functions may lead to novel diagnostic and therapeutic approaches in cancer.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

血红素氧合酶-1参与心肌细胞延迟预适应

目的:探讨血红素氧合酶-1(HO-1)在心肌细胞缺血预适应(IPC)中的作用。方法:实验分5组:正常对照组(CN组)、缺血/再灌注组(I/R组)、缺血预适应+缺血/再灌注组(PC组)、ZnPP(HO-1抑制剂)+缺血预适应+缺血/再灌注组(ZP组)和Hemin(HO-1诱导剂)+缺血/再灌注组(HE组)。测定HO-1mRNA表达、心肌细胞存活率、胞内[Ca2+]i和细胞培养液中MDA含量等。结果:PC组和HE组HO-1mRNA表达量和细胞存活率显著高于I/R组,而MDA含量和胞内[Ca2+]i则皆显著低于I/R组,PC组各指标与ZP组间有显著性差异,与HE组66较未见显著性差异。结论:IPC可以诱导HO-1mRNA表达,对心肌细胞缺血/再灌注损伤产生延迟保护作用。     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

How does the host-specialized aphid deal with food deficiency？

Aphis gossypii Glover shows obvious host specialization, with cucurbit- and cotton-specialized biotypes or host races in many regions. Because its annual natal hostcrops senesce earlier the cucurbit-specialized biotype may suffer food deficiency. The method this biotype uses to overcome this challenge is still poorly understood. In orderto understand the potential of the cucurbit-specialized biotype aphids in host shift and usage, the performance of this biotype on cotton （Gossypium hirsutum）, a common butpoor quality host plant, was explored in this study. The cucurbit-specialized aphids could establish populations on cotton only when these plants had at least nine leaves, and subsequent populations developed rather slowly. The presence of whitefly populations on cotton improved the success rate of cucurbit-specialized aphids. The cucurbit-specialized aphidswere mainly distributed on the older leaves of cotton, with only a few settling on the upper leaves. The cucurbit-specialized aphids reared on cotton for 40, 54 and 61 days stillmaintained strong preference for their natal host plant, cucumber （Cucumis sativus）, rather than cotton, and their net reproductive rates and intrinsic rates of natural increase weredramatically lower when they were transferred onto new six-leaf cotton plants or detached leaves. Therefore, we concluded that the cucurbit-specialized aphids have the potentialto utilize mature or whitefly-stressed cotton plants, but that this feeding experience on cotton did not alter their specialization for cucurbits. Some cotton plants could act as atemporary host for the cucurbit-specialized aphids to overcome food deficiency arising from senescing cucurbits.     

Plastid Signals and the Bundle Sheath： Mesophyll Development in Reticulate Mutants

The development of a plant leaf is a meticulously orchestrated sequence of events producing a complex organ comprising diverse cell types. The reticulate class of leaf variegation mutants displays contrasting pigmentation between veins and interveinal regions due to specific aberrations in the development of mesophyll cells. Thus, the reticulate mutants offer a potent tool to investigate cell-type-specific developmental processes. The discovery that most mutants are affected in plastid-localized, metabolic pathways that are strongly expressed in vasculature-associated tis- sues implicates a crucial role for the bundle sheath and their chloroplasts in proper development of the mesophyll cells. Here, we review the reticulate mutants and their phenotypic characteristics, with a focus on those in Arabidopsis thali- ana. Two alternative models have been put forward to explain the relationship between plastid metabolism and meso- phyll cell development, which we call here the supply and the signaling hypotheses. We critically assess these proposed models and discuss their implications for leaf development and bundle sheath function in C3 species. The characteriza- tion of the reticulate mutants supports the significance of plastid retrograde signaling in cell development and highlights the significance of the bundle sheath in C3 photosynthesis.