高原习服及习服不良过程中基因表达特征及其病理生理学意义研究
发布时间：2018-09-12 14:21
【摘要】：研究背景:高原地区以低氧、低气压、寒冷、相对湿度低和太阳辐射强为主要特征,其中对人类生命活动影响最为显著的主要是低氧因素。人体暴露于高原低氧环境后,机体会从整体水平调动其内在机制发生一系列的代偿适应性变化以应对高原低氧环境,使机体内环境由不平衡到平衡,最终达到内、外环境的统一,这一过程称为高原习服。机体对高原环境的习服是一个时间依赖的渐进过程,在高原低氧暴露的不同时间阶段,机体习服的优势反应不同,表明不同习服阶段的内在机制及其分子基础可能不同。多数人可通过习服获得对高原环境良好的适应,但也有部分人因对高原环境的习服不良而发生各种急、慢性高原病。急性高原病包括急性高山病(acute mountain sickness,AMS)、高原肺水肿和高原脑水肿,其中AMS是快速进入高原人群中最常见的疾病,未采取预防措施的急进高原人群AMS发病率中位值高达60%。AMS患者常常丧失正常的作业能力,重度AMS患者如未及时治疗,极易发展为高原脑水肿,直接威胁患者的生命。AMS已成为急进高原人群生命健康的主要威胁。慢性高原病包括高原红细胞增多症(high altitude polycythemia,HAPC)和高原肺动脉高压,其中HAPC是常驻高原人群中最为多见的慢性高原病,以红细胞过度增生、血红蛋白浓度显著增加为主要特征,由于红细胞过度增多导致血液粘滞度增高、微循环障碍,进一步加重组织缺氧,患者易出现全身多个器官、系统损伤和血栓等严重并发症。严重影响患者健康和生命安全。久居高原者返回平原后,随着低氧刺激的消失,机体为适应高原环境而发生的一系列功能、代谢和形态学方面的改变又要重新调整,以适应平原环境,这一过程称为脱习服。在此过程中,部分个体可表现出嗜睡、反应力和记忆力减退等一系列临床症状。有关高原习服及习服不良(高原病)的机制历来是高原医学关注的重点,虽然以往从生理、生化和形态学角度对高原习服进行了大量研究,丰富了人们对高原习服的认识,但迄今高原习服的分子机制尚不十分清楚并缺乏系统性的研究。有关AMS和HAPC发病机制的前期研究表明,AMS和HAPC的发生是众多基因以及基因与高原低氧环境相互作用的结果,因此需要从系统的角度出发对其发生发展和运转机制进行研究。基因表达变化是转录组学研究的核心内容,细胞转录组具有时空特异性,随时间和空间的改变而改变,因此基因表达变化是细胞维持稳态和改变自身状态的主要途径。对基因表达进行分析是研究复杂表型分子机制的重要研究手段,且转录组学产生的富含大量生物学信息的基因表达数据为系统性的研究复杂表型的分子基础提供了可能。因此本文围绕“高原习服及习服不良过程中基因表达特征及其病理生理学意义”这一主题,从两个部分详细开展研究。第一部分,对汉族人群进入高原前、进入高原早期、中期、后期以及返回平原早期和后期进行了连续动态的观察。在此期间,采集个体生理数据及临床症状表型,使用RNA-Seq技术检测全血细胞基因表达情况,采用WGCNA算法寻找各时间阶段特异表达基因,结合基因注释数据库以及文献对其病理生理学意义进行研究,深入了解高原习服的本质。第二部分,比较AMS和HAPC患者与未患病者高原低氧暴露后基因表达变化,构建并比较AMS和HAPC患者和未患病者差异表达基因共表达网络,从网络水平研究与AMS和HAPC发生相关的基因共表达模式及其关键基因,为高原病发病机制的研究提供重要的线索。方法:1.本研究分别进行了2次独立的人群实验:(1)动态观察16名健康男性汉族志愿者人群进入高原前、进入高原(5300m)早期(3d)、中期(4m)、后期(1y)以及返回平原早期(1m)和后期(6m)血压、心率及血氧饱和度变化,同时采集静脉血用于RNA-Seq测序、血红蛋白浓度检测以及血浆细胞因子检测;(2)采集109名健康男性汉族志愿者进入高原前的静脉血,分离血浆用于microRNA表达谱测定,观察记录志愿者进入高原(3658m)后AMS的发病情况及症状评分。2.提取全血细胞总RNA,构建PE测序文库,采用Illumina HiSeq 2000测序平台进行RNA-Seq测序。3.采用BGI标准数据处理流程评估RNA-Seq原始序列数据质量并比对到人类参考基因组(hg19);采用FPKM值对基因表达值进行标准化和定量,R软件包edgeR分析组间差异表达基因,取|Fold change|≥2且FDR≤0.05为阈值;采用WGCNA算法对基因表达量进行系统分析,根据基因表达相似性划分基因模块,获取进入高原早期、中期、后期以及返回平原早期及后期特异表达基因并根据基因间关联度构建各自共表达网络;使用clusterProfiler软件包及在线分析工具REVIGO对各阶段特异表达基因进行生物学注释;结合已报道文献对网络中关键基因的生物学功能进行详细探讨。4.使用RPKM标准化和定量AMS患者与未患病者转录本表达量;采用density based pruning算法筛选AMS患者和未患病者急进高原前后差异表达转录本;使用DAVID中Gene Functional Classification工具对差异表达转录本进行生物学注释并归类;根据聚类网络理论计算AMS患者与未发病者进入高原后基因的拓扑重合矩阵OT(i,j),并构建比较两者基因共表达网络的拓扑结构。5.使用ELISA法对AMS患者和未患病者血浆中IL10、CCL8及IL17F含量进行检测;采用化学发光法对AMS患者和未患病者IL10含量进行重复性验证。6.使用miRCURYTM LNA Array检测健康男性汉族志愿者进入高原前血浆中microRNA表达谱;使用qRT-PCR法对差异microRNA进行验证并使用R软件包OptimalCutpoints对其在AMS发病风险中的预测效能进行评价。7.使用microT-CDS和TarBase工具对microRNA的调控靶基因进行分析;使用DIANA-miRPath对microRNA靶基因进行生物学注释。8.采用FPKM对HAPC患者和未发病者基因表达标准化和定量,R软件包egdeR分析组间差异表达基因,取|Fold change|≥2且FDR≤0.05为阈值;使用clusterProfiler软件包及在线分析工具REVIGO对HAPC患者和未发病者关联基因进行生物学注释;采用斯皮尔曼相关系数法构建HAPC患者和未患病者进入高原后差异上调基因共表达网络;采用Concentric对HAPC患者和未患病者基因共表达网络进行比较,明确HAPC发生相关的基因共表达模式及关键基因。结果:1.进入高原早期,参与多巴胺代谢、离子转运及血红蛋白合成的基因特异性表达,基因MXI1、RNF10、TRIM58、GLRX5和BPGM处于共表达网络的中心位置;中期,基因表达模式与进入高原前相似;后期,参与磷代谢、免疫系统与MAPK信号通路调节、细胞内吞及囊泡转运的基因特异性表达,基因SMARCD2、CDK9及RIC8A处于共表达网络的中心位置。2.返回平原早期,参与大分子物质、核酸代谢,细胞增殖分化的基因特异性表达,基因MTF2、ZFR、CAND1、DEPDC1、DEPDC1B、CCNA2、CDC6、CDC20、CCNB2、CCNB1和ANLN处于共表达网络的中心位置;后期参与免疫炎症反应的基因特异性表达,基因ZBP1、STAT2、IFIT1、IFIT2和IFIT3处于共表达网络的中心位置。3.与AMS未发病者相比,免疫和炎症反应在AMS患者差异表达转录本中显著富集(富集分数:3.44,3.27);AMS患者与未患病者基因共表达网络中IL10、CCL8和IL17F的度存在显著差异;蛋白水平上,进入高原后AMS患者IL10的表达显著下调,CCL8和IL17F显著增加,未发病者IL10显著上调,IL17F显著下调,CCL8改变无统计学差异;且在另一人群中,急进高原后,AMS患者IL10蛋白显著下调(p=0.001),未患病者无显著改变,IL10的改变量与急进高原个体临床症状评分间存在较强的相关性,r(22)=-0.52,p=0.013。4.进入高原前,AMS患者血浆miR-369-3p、miR-449b-3p和miR-136-3p表达量显著高于未患病者;采用Logistic回归模型发现miR-369-3p、miR-449b-3p和miR-136-3p组合对AMS发病风险预测的敏感度和特异度达到92.68%和93.48%,AUC:0.986,95%CI:0.970-1.000,p0.001,LR+:14.21,LR-:0.08;生物信息学分析提示miR-369-3p,mi R-449b-3p和mi R-136-3p调控的靶基因主要参与含氮化合物的代谢过程及神经营养因子TRK受体信号通路。5.HAPC患者较未发病者具有311个特有的差异上调基因,显著富集于细胞增殖以及免疫调控过程;进入高原后,HAPC患者表达上调基因共表达网络平均度为22.64,是未患病者的2倍(11.85);HAPC患者差异上调基因共表达网络中,中心性(Betweeness centrality)较高的基因为IFT20和MRPL22,分别为0.50和0.46,未患病者中,中心性较高的基因为ITGAV和TPRKB,分别为0.50和0.37。结论:1.进入高原早期,机体以MXI1、RNF10、TRIM58、GLRX5及BPGM为核心表达基因,调控参与多巴胺代谢、离子转运及血红蛋白合成基因,提示在高原习服早期,机体主要通过增加氧气摄入、运输以及氧合血红蛋白氧释放效率等适应高原环境,以实现机体内、外环境的基本平衡;随着高原暴露时间的延长,机体调动更多内在机制参与内环境稳态的调节,其中以SMARCD2、CDK9及RIC8A为核心表达基因。这些基因通过调控参与磷代谢、免疫系统与MAPK信号通路调节、细胞内吞及囊泡转运的基因,促进红细胞增殖增加氧的运输,促进组织血管新生缩短氧的弥散距离以及增强组织细胞对氧的利用等多种机制习服高原环境。在此过程中,机体的免疫系统激活,其意义可能在于防御低氧损伤,确保机体自身稳定。2.从高原返回平原早期,机体以MTF2、DEPDC1、DEPDC1B、CCNA2、CDC6、CDC20、CCNB2、CCNB1和ANLN为核心表达基因,通过调控细胞的增殖分化,建立内环境平衡状态;后期以ZBP1、STAT2及IFIT家族为核心表达基因,参与并增强免疫炎症反应,清除内源性损伤因子并修复损伤组织。3.炎症反应增强是AMS发生的重要机制。高原低氧引起AMS患者基因共表达模式改变,导致抗炎因子IL10分泌减少,促炎细胞因子CCL8和IL17F分泌增加,可能是促使炎症反应增强进而发生AMS的重要机制。4.汉族人进入高原前的血浆microRNA水平与AMS的发病风险密切相关,其中血浆miR-369-3p、miR-449b-3p和miR-136-3p分子组合对AMS的发病风险具有很好的预测效能,提示其是一种新的预测AMS易感性的生物标志物。5.参与细胞增殖以及免疫反应的基因过度上调以及基因间特有的共表达关系与HAPC的发生密切相关,其中以IFT20和MRPL22的作用尤为关键,提示其可能是HAPC发病的重要环节和防治的重要靶点。综上所述,本论文采用RNA-Seq技术首次对汉族人群进入高原前,进入高原早期、中期和后期以及返回平原早期和后期全过程全血细胞基因表达进行了动态观察,从系统的角度对高原习服及脱习服过程中不同时间段特异表达基因及其病理生理学意义进行了研究,为理解高原习服的本质提供了重要的理论依据。从整体水平对AMS和HAPC患者与未患病者的基因表达进行了分析比较,发现了与AMS和HAPC发病相关的基因共表达模式及其关键基因,为今后深入研究AMS和HAPC提供了重要的线索。同时,首次报导了循环microRNA分子在AMS的发病风险预测中的作用,发现了可用于AMS预防的全新的生物标志物。
[Abstract]:BACKGROUND: The plateau area is characterized by hypoxia, low pressure, cold, low relative humidity and strong solar radiation. The most significant factors affecting human life activities are hypoxia. High altitude acclimatization is a time-dependent gradual process in which the body acclimatizes to the high altitude hypoxic environment. At different stages of altitude hypoxic exposure, the body acclimatizes to different advantages, indicating different acclimatization stages. The underlying mechanism and molecular basis may be different. Most people can acquire a good adaptation to the high altitude environment by acclimation, but some people develop various acute and chronic high altitude diseases due to poor acclimation to the high altitude environment. Acute high altitude diseases include acute mountain sickness (AMS), high altitude pulmonary edema and high altitude brain edema, including AMS. AMS is the most common disease among people who enter the plateau rapidly. The median incidence of AMS is as high as 60% among people who enter the plateau rapidly without preventive measures. AMS patients often lose their normal working ability. If severe AMS patients are not treated in time, they will easily develop into high altitude brain edema, which directly threatens the lives of patients. AMS has become a healthy life for people who enter the plateau rapidly. Chronic plateau diseases include high altitude polycythemia (HAPC) and high altitude pulmonary hypertension, of which HAPC is the most common chronic plateau disease in people living at high altitudes, characterized by excessive erythrocyte hyperplasia and marked increase in hemoglobin concentration, resulting in excessive red blood cell hyperplasia leading to blood pressure. High viscosity, microcirculation disturbance, and further aggravation of tissue hypoxia, patients are prone to multiple organs of the body, system damage and thrombosis and other serious complications, seriously affecting the health and safety of patients. Morphological changes have to be readjusted to fit the plain environment, a process known as acclimatization, in which some individuals exhibit a range of clinical symptoms such as drowsiness, loss of responsiveness and memory. Physiological, biochemical and morphological studies on altitude acclimatization have enriched people's understanding of altitude acclimatization, but the molecular mechanism of altitude acclimatization is still unclear and lack of systematic research.Previous studies on the pathogenesis of AMS and HAPC have shown that the occurrence of AMS and HAPC is a large number of genes, as well as genes and altitude lows. Gene expression changes are the core of transcriptome research. Cell transcriptome is space-time specific and changes with time and space. Therefore, gene expression changes are the maintenance of homeostasis and self-change of cells. The analysis of gene expression is an important means to study the molecular mechanism of complex phenotypes, and the data of gene expression rich in biological information produced by transcriptome provide a possibility for the systematic study of the molecular basis of complex phenotypes. In the first part, a continuous dynamic observation was carried out on the Han population before entering the plateau, early, middle, late, and early and late return to the plain. RNA-Seq technique was used to detect gene expression in whole blood cells, and WGCNA algorithm was used to find the specific expression genes at different time stages. The pathophysiological significance of gene annotation database and literature were studied to understand the essence of altitude acclimation. Part 2: Comparing AMS and HAPC patients with and without altitude hypoxia exposure. The co-expression patterns and key genes related to the occurrence of AMS and HAPC were studied from the network level, providing important clues for the study of the pathogenesis of altitude sickness. Methods: 1. Two independent population studies were conducted. The results were as follows: (1) Blood pressure, heart rate and oxygen saturation were dynamically observed in 16 healthy male Han volunteers before entering the plateau, at the early (3d), middle (4m), late (1y) and early (1m) and late (6m) after returning to the plateau, and venous blood was collected for RNA-Seq sequencing, hemoglobin concentration detection and plasma cytokines detection. (2) Blood samples were collected from 109 healthy male Han volunteers before they entered the plateau. Plasma samples were separated for microRNA expression profiling. The incidence and symptom score of AMS were observed and recorded after they entered the plateau (3658m). Total RNA of whole blood cells was extracted and the PE sequencing library was constructed. RNA-Seq was sequenced by Illumina HiSeq 2000. The quality of original RNA-Seq sequence data was assessed by BGI standard data processing flow and compared with human reference genome (hg19); the gene expression values were standardized and quantified by FPKM value; the differentially expressed genes between groups were analyzed by R software package edgeR, and the | Fold change | 2 and FDR | 0.05 were taken as thresholds; the gene expression was systematically analyzed by WGCNA algorithm. According to the similarity of gene expression, we divided the gene modules into early, middle, late and return to the plain, and constructed their co-expression networks according to the degree of gene association; using cluster Profiler software package and on-line analysis tool REVIGO to biologically analyze the stage-specific genes. Notes: Biological functions of key genes in the network were discussed in detail with the reported literature. 4. RPKM was used to standardize and quantify the transcripts of AMS patients and non-AMS patients; density-based pruning algorithm was used to screen differentially expressed transcripts of AMS patients and non-AMS patients before and after acute plateau entry; and Gene Functional Clas in DAVID was used. The sification tool was used to annotate and classify the differentially expressed transcripts; the topological coincidence matrix OT (i, j) of AMS patients and non-AMS patients was calculated according to clustering network theory, and the topological structure of the gene co-expression network was constructed and compared. 5. The plasma IL 10, CCL8 and IL in AMS patients and non-AMS patients were analyzed by ELISA. Serum microRNA expression profiles of healthy male Han volunteers before entering the plateau were detected by using microRNAs microRNA expression profiles; differentially expressed microRNAs were validated by qRT-PCR and were detected by R software package Optimal Cutpoints in AMS. Evaluation of predictive efficacy in disease risk. 7. MicroRNA regulatory target genes were analyzed using microT-CDS and TarBase tools; microRNA target genes were biologically annotated using DIANA-MicroPath. Old change | 2 and FDR < 0.05 were used as thresholds; the association genes between HAPC patients and non-HAPC patients were biologically annotated by cluster Profiler software package and on-line analysis tool REVIGO; the co-expression networks of differentially up-regulated genes between HAPC patients and non-HAPC patients were constructed by Spearman correlation coefficient method; and the concentric was used for HAPC patients. Results: 1. In the early stage of Plateau entry, genes involved in dopamine metabolism, ion transport and hemoglobin synthesis were specifically expressed. Genes MXI1, RNF10, TRIM58, GLRX5 and BPGM were at the center of the co-expression network. The gene expression pattern was similar to that before entering the plateau; at the later stage, the genes involved in phosphorus metabolism, immune system and MAPK signaling pathway, endocytosis and vesicle transport were specifically expressed, and SMARCD2, CDK9 and RIC8A were at the center of the co-expression network. 2. Back to the early plain, the genes involved in macromolecular substances, nucleic acid metabolism, cell proliferation and differentiation. Because of specific expression, genes MTF2, ZFR, CAND1, DEPDC1, DEPDC1B, CCNA2, CDC6, CDC20, CCNB2, CCNB1 and ANLN are at the center of the co-expression network; genes ZBP1, STAT2, IFIT1, IFIT2 and IFIT3 are at the center of the co-expression network in the late stage of immune inflammation, compared with those without AMS. The response was significantly enriched in the differentially expressed transcripts of AMS patients (enrichment fraction: 3.44, 3.27); the degree of IL 10, CCL8 and IL 17F in the gene co-expression network of AMS patients and non-AMS patients was significantly different; at protein level, the expression of IL 10 in AMS patients was significantly down-regulated, CCL8 and IL 17F were significantly increased, IL 10 was significantly up-regulated in non-AMS patients, and IL 17F was significantly up-regulated in AMS patients at high altitude. There was no significant difference in CCL8, and in another group, IL-10 protein was significantly decreased in AMS patients (p = 0.001) after acute high altitude entry, and no significant change was found in non-AMS patients. There was a strong correlation between the change of IL-10 and the individual clinical symptom score of acute high altitude entry, R (22) = - 0.52, P = 0.013.4. The expression levels of 9b-3p and microwave-136-3p were significantly higher than those of non-patients; Logistic regression model showed that the sensitivity and specificity of the combination of microwave-369-3p, microwave-449b-3p and microwave-136-3p to the risk prediction of AMS were 92.68% and 93.48%, AUC: 0.986, 95% CI: 0.970-1.000, p0.001, LR +: 14.21, LR - - 0.08; Bioinformatics analysis indicated that the combination of microwave-369-3p, microwave-449-3b, and microwave-449b-3p was sensitive and specific to the risk prediction of AMS. The target genes regulated by MIR-136-3p and MIR-136-3p are mainly involved in the metabolic process of nitrogen compounds and the signaling pathway of neurotrophic factor TRK receptor. The mean value was 22.64, twice as high as that of non-HAPC patients (11.85). In the co-expression network of differentially up-regulated genes in HAPC patients, the genes with higher centrality were IFT2 0 and MRPL22, respectively, 0.50 and 0.46. In non-HAPC patients, the genes with higher centrality were ITGAV and TPRKB, respectively, 0.50 and 0.37. Conclusion: 1. In the early stage of Plateau entry, the organisms with MXI1, R, and TPRKB had higher centrality. NF10, TRIM58, GLRX5 and BPGM are the core expression genes, which regulate dopamine metabolism, ion transport and hemoglobin synthesis. It is suggested that in the early stage of altitude acclimation, the organism adapts to altitude environment mainly by increasing oxygen intake, transport and oxygen release efficiency of oxyhemoglobin, so as to achieve the basic balance between the internal and external environment. With prolonged exposure time, the body mobilizes more internal mechanisms to participate in the regulation of homeostasis of the internal environment, in which SMARCD2, CDK9 and RIC8A are the core expression genes. These genes are involved in phosphorus metabolism, immune system and MAPK signal pathway regulation, endocytosis and vesicle transport genes, promote red blood cell proliferation and oxygen transport, promote Tissue angiogenesis shortens the diffusion distance of oxygen and enhances the utilization of oxygen by tissue cells. During this process, the immune system is activated, which may be of significance in preventing hypoxic injury and ensuring the stability of the body. 2. In the early stage of returning to the plain from the plateau, the body uses MTF2, DEPDC1, DEPDC1B, CCNA2, CDC6, CDC20. CCNB2, CCNB1 and ANLN are the core expression genes, which regulate cell proliferation and differentiation to establish internal environment balance; ZBP1, STAT2 and IFIT family are the core expression genes in the late stage, participate in and enhance immune inflammation, remove endogenous injury factors and repair damaged tissues. 3. Increased inflammation is an important mechanism of AMS. Oxygen induces alterations in gene co-expression patterns in AMS patients, resulting in decreased secretion of anti-inflammatory factors IL10 and increased secretion of pro-inflammatory cytokines CCL8 and IL17F.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R594.3
本文编号：2239294
[Abstract]:BACKGROUND: The plateau area is characterized by hypoxia, low pressure, cold, low relative humidity and strong solar radiation. The most significant factors affecting human life activities are hypoxia. High altitude acclimatization is a time-dependent gradual process in which the body acclimatizes to the high altitude hypoxic environment. At different stages of altitude hypoxic exposure, the body acclimatizes to different advantages, indicating different acclimatization stages. The underlying mechanism and molecular basis may be different. Most people can acquire a good adaptation to the high altitude environment by acclimation, but some people develop various acute and chronic high altitude diseases due to poor acclimation to the high altitude environment. Acute high altitude diseases include acute mountain sickness (AMS), high altitude pulmonary edema and high altitude brain edema, including AMS. AMS is the most common disease among people who enter the plateau rapidly. The median incidence of AMS is as high as 60% among people who enter the plateau rapidly without preventive measures. AMS patients often lose their normal working ability. If severe AMS patients are not treated in time, they will easily develop into high altitude brain edema, which directly threatens the lives of patients. AMS has become a healthy life for people who enter the plateau rapidly. Chronic plateau diseases include high altitude polycythemia (HAPC) and high altitude pulmonary hypertension, of which HAPC is the most common chronic plateau disease in people living at high altitudes, characterized by excessive erythrocyte hyperplasia and marked increase in hemoglobin concentration, resulting in excessive red blood cell hyperplasia leading to blood pressure. High viscosity, microcirculation disturbance, and further aggravation of tissue hypoxia, patients are prone to multiple organs of the body, system damage and thrombosis and other serious complications, seriously affecting the health and safety of patients. Morphological changes have to be readjusted to fit the plain environment, a process known as acclimatization, in which some individuals exhibit a range of clinical symptoms such as drowsiness, loss of responsiveness and memory. Physiological, biochemical and morphological studies on altitude acclimatization have enriched people's understanding of altitude acclimatization, but the molecular mechanism of altitude acclimatization is still unclear and lack of systematic research.Previous studies on the pathogenesis of AMS and HAPC have shown that the occurrence of AMS and HAPC is a large number of genes, as well as genes and altitude lows. Gene expression changes are the core of transcriptome research. Cell transcriptome is space-time specific and changes with time and space. Therefore, gene expression changes are the maintenance of homeostasis and self-change of cells. The analysis of gene expression is an important means to study the molecular mechanism of complex phenotypes, and the data of gene expression rich in biological information produced by transcriptome provide a possibility for the systematic study of the molecular basis of complex phenotypes. In the first part, a continuous dynamic observation was carried out on the Han population before entering the plateau, early, middle, late, and early and late return to the plain. RNA-Seq technique was used to detect gene expression in whole blood cells, and WGCNA algorithm was used to find the specific expression genes at different time stages. The pathophysiological significance of gene annotation database and literature were studied to understand the essence of altitude acclimation. Part 2: Comparing AMS and HAPC patients with and without altitude hypoxia exposure. The co-expression patterns and key genes related to the occurrence of AMS and HAPC were studied from the network level, providing important clues for the study of the pathogenesis of altitude sickness. Methods: 1. Two independent population studies were conducted. The results were as follows: (1) Blood pressure, heart rate and oxygen saturation were dynamically observed in 16 healthy male Han volunteers before entering the plateau, at the early (3d), middle (4m), late (1y) and early (1m) and late (6m) after returning to the plateau, and venous blood was collected for RNA-Seq sequencing, hemoglobin concentration detection and plasma cytokines detection. (2) Blood samples were collected from 109 healthy male Han volunteers before they entered the plateau. Plasma samples were separated for microRNA expression profiling. The incidence and symptom score of AMS were observed and recorded after they entered the plateau (3658m). Total RNA of whole blood cells was extracted and the PE sequencing library was constructed. RNA-Seq was sequenced by Illumina HiSeq 2000. The quality of original RNA-Seq sequence data was assessed by BGI standard data processing flow and compared with human reference genome (hg19); the gene expression values were standardized and quantified by FPKM value; the differentially expressed genes between groups were analyzed by R software package edgeR, and the | Fold change | 2 and FDR | 0.05 were taken as thresholds; the gene expression was systematically analyzed by WGCNA algorithm. According to the similarity of gene expression, we divided the gene modules into early, middle, late and return to the plain, and constructed their co-expression networks according to the degree of gene association; using cluster Profiler software package and on-line analysis tool REVIGO to biologically analyze the stage-specific genes. Notes: Biological functions of key genes in the network were discussed in detail with the reported literature. 4. RPKM was used to standardize and quantify the transcripts of AMS patients and non-AMS patients; density-based pruning algorithm was used to screen differentially expressed transcripts of AMS patients and non-AMS patients before and after acute plateau entry; and Gene Functional Clas in DAVID was used. The sification tool was used to annotate and classify the differentially expressed transcripts; the topological coincidence matrix OT (i, j) of AMS patients and non-AMS patients was calculated according to clustering network theory, and the topological structure of the gene co-expression network was constructed and compared. 5. The plasma IL 10, CCL8 and IL in AMS patients and non-AMS patients were analyzed by ELISA. Serum microRNA expression profiles of healthy male Han volunteers before entering the plateau were detected by using microRNAs microRNA expression profiles; differentially expressed microRNAs were validated by qRT-PCR and were detected by R software package Optimal Cutpoints in AMS. Evaluation of predictive efficacy in disease risk. 7. MicroRNA regulatory target genes were analyzed using microT-CDS and TarBase tools; microRNA target genes were biologically annotated using DIANA-MicroPath. Old change | 2 and FDR < 0.05 were used as thresholds; the association genes between HAPC patients and non-HAPC patients were biologically annotated by cluster Profiler software package and on-line analysis tool REVIGO; the co-expression networks of differentially up-regulated genes between HAPC patients and non-HAPC patients were constructed by Spearman correlation coefficient method; and the concentric was used for HAPC patients. Results: 1. In the early stage of Plateau entry, genes involved in dopamine metabolism, ion transport and hemoglobin synthesis were specifically expressed. Genes MXI1, RNF10, TRIM58, GLRX5 and BPGM were at the center of the co-expression network. The gene expression pattern was similar to that before entering the plateau; at the later stage, the genes involved in phosphorus metabolism, immune system and MAPK signaling pathway, endocytosis and vesicle transport were specifically expressed, and SMARCD2, CDK9 and RIC8A were at the center of the co-expression network. 2. Back to the early plain, the genes involved in macromolecular substances, nucleic acid metabolism, cell proliferation and differentiation. Because of specific expression, genes MTF2, ZFR, CAND1, DEPDC1, DEPDC1B, CCNA2, CDC6, CDC20, CCNB2, CCNB1 and ANLN are at the center of the co-expression network; genes ZBP1, STAT2, IFIT1, IFIT2 and IFIT3 are at the center of the co-expression network in the late stage of immune inflammation, compared with those without AMS. The response was significantly enriched in the differentially expressed transcripts of AMS patients (enrichment fraction: 3.44, 3.27); the degree of IL 10, CCL8 and IL 17F in the gene co-expression network of AMS patients and non-AMS patients was significantly different; at protein level, the expression of IL 10 in AMS patients was significantly down-regulated, CCL8 and IL 17F were significantly increased, IL 10 was significantly up-regulated in non-AMS patients, and IL 17F was significantly up-regulated in AMS patients at high altitude. There was no significant difference in CCL8, and in another group, IL-10 protein was significantly decreased in AMS patients (p = 0.001) after acute high altitude entry, and no significant change was found in non-AMS patients. There was a strong correlation between the change of IL-10 and the individual clinical symptom score of acute high altitude entry, R (22) = - 0.52, P = 0.013.4. The expression levels of 9b-3p and microwave-136-3p were significantly higher than those of non-patients; Logistic regression model showed that the sensitivity and specificity of the combination of microwave-369-3p, microwave-449b-3p and microwave-136-3p to the risk prediction of AMS were 92.68% and 93.48%, AUC: 0.986, 95% CI: 0.970-1.000, p0.001, LR +: 14.21, LR - - 0.08; Bioinformatics analysis indicated that the combination of microwave-369-3p, microwave-449-3b, and microwave-449b-3p was sensitive and specific to the risk prediction of AMS. The target genes regulated by MIR-136-3p and MIR-136-3p are mainly involved in the metabolic process of nitrogen compounds and the signaling pathway of neurotrophic factor TRK receptor. The mean value was 22.64, twice as high as that of non-HAPC patients (11.85). In the co-expression network of differentially up-regulated genes in HAPC patients, the genes with higher centrality were IFT2 0 and MRPL22, respectively, 0.50 and 0.46. In non-HAPC patients, the genes with higher centrality were ITGAV and TPRKB, respectively, 0.50 and 0.37. Conclusion: 1. In the early stage of Plateau entry, the organisms with MXI1, R, and TPRKB had higher centrality. NF10, TRIM58, GLRX5 and BPGM are the core expression genes, which regulate dopamine metabolism, ion transport and hemoglobin synthesis. It is suggested that in the early stage of altitude acclimation, the organism adapts to altitude environment mainly by increasing oxygen intake, transport and oxygen release efficiency of oxyhemoglobin, so as to achieve the basic balance between the internal and external environment. With prolonged exposure time, the body mobilizes more internal mechanisms to participate in the regulation of homeostasis of the internal environment, in which SMARCD2, CDK9 and RIC8A are the core expression genes. These genes are involved in phosphorus metabolism, immune system and MAPK signal pathway regulation, endocytosis and vesicle transport genes, promote red blood cell proliferation and oxygen transport, promote Tissue angiogenesis shortens the diffusion distance of oxygen and enhances the utilization of oxygen by tissue cells. During this process, the immune system is activated, which may be of significance in preventing hypoxic injury and ensuring the stability of the body. 2. In the early stage of returning to the plain from the plateau, the body uses MTF2, DEPDC1, DEPDC1B, CCNA2, CDC6, CDC20. CCNB2, CCNB1 and ANLN are the core expression genes, which regulate cell proliferation and differentiation to establish internal environment balance; ZBP1, STAT2 and IFIT family are the core expression genes in the late stage, participate in and enhance immune inflammation, remove endogenous injury factors and repair damaged tissues. 3. Increased inflammation is an important mechanism of AMS. Oxygen induces alterations in gene co-expression patterns in AMS patients, resulting in decreased secretion of anti-inflammatory factors IL10 and increased secretion of pro-inflammatory cytokines CCL8 and IL17F.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R594.3
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