【作者】 郭敏；

【导师】 黄盛文；

【作者基本信息】 贵州大学 ， 生物技术与工程， 2025， 硕士

【摘要】 单细胞测序(Single-Cell Sequencing,SCS)是对单个细胞的基因组或转录组进行测序分析的技术,目前已被广泛应用到基础生物研究和临床医学研究。早前研究中对有核红细胞大类进行分析,并监测其在不同状态下的变化,从而深入理解有核红细胞在发育、疾病等过程中的作用和调控机制,并探寻每类亚群之间的差异以及联系。本研究通过通过生物信息学方法分析基于单细胞测序技术的孕中晚期不同孕周(32、36、40周)脐带血有核红细胞转录组信息,对有核红细胞进行亚群注释、差异基因分析、GO/KEGG(Gene Ontology/Kyoto Encyclopedia of Genes and Genomes)功能分析以及转录因子分析,获得有核红细胞亚群的动态发育的相关基础信息,为进一步探明有核红细胞发育和分化分子机制提供新的研究靶点。主要研究内容如下:1、有核红细胞亚群注释结果:三组不同孕周(28≤孕周≤32周、孕周36周以及孕周≥40周)9例样本一共得到17个Cluster,共六类细胞群,分别是有核红细胞、B细胞、T细胞、NK细胞、单核细胞以及巨核细胞。在有核红细胞类群中鉴定出14个Cluster,11个细胞亚群,除了传统分类的原始红细胞(Proerythroblasts,Pro E)、早幼红细胞(Basophilicerythroblasts,BaosE)、中幼红细胞(Polychromatophilicerythroblasts,Ploy E)和晚幼红细胞(Orthochromaticerythoblasts,Ortho E)四个亚群外,发现无经典标志基因的Cluster,我们采用了细胞类群中差异显著且高表达的基因进行注释命名的类群分别是C1-HBD((δ-珠蛋白)、C5-TPT1((译控制蛋白)、C12-RPS10((核体蛋蛋白),最后对无marker基因和差异显著基因的Cluster,我们利用数字进行注释命名的类群分别是C0、C2、C3、C8。2、有核红细胞亚群中差异基因在GO/KEGG的富集情况:对有核红细胞亚群中类群进行差异基因筛选其中差异基因主要集中在36vs32((下调)组有967个,40vs36((上调)组有1145个,另外四组36vs32((上调)、40vs32((下调)、40vs32(上调)、40vs36((下调)中差异基因则相对较少分别是289、198、167、159个,连续变化的基因一共99个。在36vs32下调/上调组中,下调基因GO富集于细胞呼吸、缺氧反应、RNA聚合酶II转录负向调控、电子传递偶合质子传递等生物过程,C5-TPT1类群下调基因富集于核体蛋小亚基组装等;上调基因与氧气和二氧化碳运输、过氧化氢分解代谢过程相关,C8类群涉及病毒防御等,C5-TPT1类群与蛋白质泛素化等有关。KEGG下调基因与B细胞受蛋信号通路、铁死亡、细胞周期相关,C8类群与钙信号通路联系,C1-HBD、C5-TPT1主要富集在核体蛋通路,上调基因则与抗原处理和呈递、吞噬蛋等通路有关。在40vs32孕周组中,365个差异表达基因显示,下调基因富集在缺氧反应和线粒蛋电子传递过程,而上调基因富集在线粒蛋ATP合成和二氧化碳运输过程。KEGG分析显示,Pro E类群中下调基因与吞噬蛋和铁死亡通路相关,而C3、C8、C2和早幼红细胞类群的上调基因与甘氨酸、丝氨酸和苏氨酸代谢以及氮代谢通路相关。C5-TPT1类群的下调基因与细胞质译、核体蛋小亚基组装和生物发生相关。40vs36下调/上调组中,下调基因主要集中在氧气、二氧化碳运输和过氧化氢分解代谢过程。KEGG分析显示,Baso E和Ploy E类群在铁死亡和线粒蛋自噬通路中显著富集,C1-HBD类群与抗原加工、呈递及IL-17信号通路相关,其他类群如Pro E和Ortho E则与间隙连接和RNA转运通路相关。上调基因在电子传递耦合质子传递、线粒蛋ATP合成等过程显著富集,并在核体蛋和氮代谢通路中发挥重要作用,其中Baso E类群的基因还与Wnt信号通路相关。经过对转录因子进行GO和KEGG富集分析发现与红系发育相关的转录因子NFE2、ZNF367和NFIA具有不同的功能,NFE2与血红蛋白生物合成、线粒蛋自噬、衰老和代谢相关;ZNF367涉及细胞周期S期、有核红细胞分化、细胞增殖和铁死亡;NFIA则调控DNA转录、RNA聚合酶II介导的转录及蛋白质磷酸化,并在Erb B信号通路、慢性髓系白血病和铁死亡中显著富集。3、有核红细胞亚群中显著表达的基因以及转录因子的Q-PCR结果:通过筛选与红系发育、分化相关且在有核红细胞亚群中显著表达的基因以及转录因子做Q-PCR实验,结果且均符合单细胞测序分析的表达趋势。在36vs32孕周组中,以32孕周组为对照,下调组筛选出SOX6、SP100等基因,其中AFF3、LYN显著下调;上调组中HBG2、ALAS2等基因表达上升,HBB基因上调明显且有显著差异。在40vs32孕周组中,以32孕周组为对照,富集的差异基因较少,下调组中TMCC2、ENSC00000286621、H19显著下调;上调组中HBD、HLA-A、HBB等基因显著上调。在40vs36孕周组中,以36孕周组为对照,下调组筛选出BNIP3L、HBZ等基因,NCOA4、YBX3、FTH1下调明显;上调组中BCL11B、PRKCH等基因显著上调,而MALAT1在36vs32孕周组中下调,在40vs36孕周组中却显著上调。另外还验证了7个连续变化基因发现SLC4A1、FECH、CA1、HBB、IFIT1B、ALAS2基因表达量持续上调,其中HBB上调趋势尤为显著;HBG1则36孕周组升高后于40孕周组下降。转录因子TCF12、NFATC3、MYBL2、EP300的表达在36孕周组下降而后在40孕周组升高,而BCL6的表达持续上升。更多还原

【Abstract】 Single-cell sequencing(SCS)is a technology for analyzing the genome or transcriptome of individual cells and is widely used in basic biological and clinical research.Earlier studies analyzed nucleated red blood cells in bulk and monitored their changes in different states to understand their roles and regulatory mechanisms in development and diseases.This study uses bioinformatics to analyze transcriptomic data from nucle-ated red blood cells in umbilical cord blood of different gestational weeks(32,36,and40 weeks)obtained via single-cell sequencing.It annotates subpopulations of nucleated red blood cells,identifies differentially expressed genes,and performs GO/KEGG functional and transcription factor analyses to uncover the dynamic development of these cells,offering new insights into the molecular mechanisms of erythrocyte development and differentiation.The main research contents are as follows:1.A total of 17 clusters across six cell types were identified from nine samples(28–32 weeks,36 weeks,and≥40 weeks):nucleated red blood cells,B cells,T cells,NK cells,monocytes,and megakaryocytes.Within the n RBC population,14 clusters and 11 subpopulations were identified.In addition to the four classic subtypes(pro-erythroblasts,basophilic erythroblasts,polychromatophilic erythroblasts,and ortho-chromatic erythroblasts),novel clusters were annotated based on differentially ex-pressed genes:C1-HBD(δ-globin),C5-TPT1(translation control protein),and C12-RPS10(ribosomal protein).Clusters without marker genes or significantly differen-tially expressed genes were annotated as C0,C2,C3,and C8.2.In the differential gene analysis of nucleated red blood cell subpopulations,the36vs32(downregulated)group had 967 DEGs,and the 40vs36(upregulated)group had1,145 DEGs.The other four groups(36vs32 upregulated,40vs32 downregulated,40vs32 upregulated,and 40vs36 downregulated)had fewer DEGs,with counts of 289,198,167,and 159,respectively.Overall,there were 99 continuously changing genes.In the 36vs32 down/upregulated group,downregulated genes were enriched in bi-ological processes like cellular respiration,hypoxic response,negative regulation of RNA Polymerase II transcription,and electron transport coupled proton transport.Downregulated genes in the C5-TPT1 cluster were enriched in ribosomal small subunit assembly.Upregulated genes were associated with oxygen and carbon dioxide transport,as well as hydrogen peroxide metabolism.The C8 cluster was involved in viral defense,and the C5-TPT1 cluster was linked to protein ubiquitination.KEGG analysis showed downregulated genes were associated with the B cell receptor signaling pathway,fer-roptosis,and cell cycle.The C8 cluster was linked to calcium signaling,while the C1-HBD and C5-TPT1 clusters were enriched in ribosome-related pathways.Upregulated genes were associated with antigen processing and presentation,and phagosome path-ways.In the 40vs32 gestational age group,365 differentially expressed genes were iden-tified.Downregulated genes were enriched in hypoxic response and mitochondrial elec-tron transport processes,while upregulated genes were enriched in mitochondrial ATP synthesis and carbon dioxide transport.KEGG analysis revealed that downregulated genes in the Pro E cluster were associated with phagosome and ferroptosis pathways.Upregulated genes in the C3,C8,C2,and early erythroblast clusters were linked to metabolic pathways of glycine,serine,threonine,and nitrogen.Downregulated genes in the C5-TPT1 cluster were associated with cytoplasmic translation,ribosomal subunit assembly,and biogenesis.In the 40vs36 down/upregulated group,downregulated genes were mainly focused on oxygen,carbon dioxide transport,and hydrogen peroxide metabolism.KEGG anal-ysis showed that Baso E and Ploy E clusters were significantly enriched in ferroptosis and mitochondrial autophagy pathways.The C1-HBD cluster was linked to antigen processing,presentation,and the IL-17 signaling pathway.Other clusters,like Pro E and Ortho E,were related to gap junction and RNA transport pathways.Upregulated genes were significantly enriched in processes like electron transport coupled proton transport and mitochondrial ATP synthesis,and played key roles in ribosomal and nitrogen met-abolic pathways.Notably,genes in the Baso E cluster were also associated with the Wnt signaling pathway.Enrichment analysis of transcription factors identified NFE2,ZNF367,and NFIA as key transcription factors associated with erythroid development,each with distinct functions.NFE2 was associated with haemoglobin biosynthesis,mitophagy,senescence,and metabolism;ZNF367 with cell-cycle S phase,erythroblast differentiation,prolifer-ation,and ferroptosis;and NFIA with DNA transcription,RNA polymerase II-mediated transcription and protein phosphorylation.NFIA was also significantly enriched in the Erb B signaling pathway,chronic myeloid leukaemia and ferroptosis.3.We conducted Q-PCR experiments on genes and transcription factors related to erythroid development and differentiation and significantly expressed in nucleated red blood cell subclusters.The results were consistent with the expression trends from sin-gle-cell sequencing analysis.In the 36vs32 gestational week group(using the 32-week group as the control),down-regulated genes such as SOX6 and SP100 were identified,with AFF3 and LYN showing significant down-regulation;in the up-regulated group,genes like HBG2 and ALAS2 exhibited a gradual increase,with HBB showing the most significant up-regu-lation.In the 40vs36 gestational week group(using the 36-week group as the control),down-regulated genes such as BNIP3L and HBZ were identified,with NCOA4,YBX3,and FTH1 showing marked down-regulation;in the up-regulated group,genes like BCL11B and PRKCH were significantly up-regulated,and MALAT1,which was down-regulated in the 36vs32 group,was significantly up-regulated here.In the 40vs32 ges-tational week group,fewer differentially expressed genes were detected.In the down-regulated group,TMCC2,ENSC00000286621,and H19 exhibited significant down-regulation;in the up-regulated group,HBD,HLA-A,HBB,etc.,showed significant up-regulation.Additionally,validation of seven continuously variable genes revealed that the expression levels of SLC4A1,FECH,CA1,and HBB showed sustained up-regulation,with HBB exhibiting the most significant upward trend.IFIT1B and ALAS2 increased at 36 weeks but decreased at 40 weeks,while HBG1 showed sustained and significant down-regulation.Among transcription factors,TCF12,NFATC3,MYBL2,and EP300 decreased at 36 weeks and then increased at 40 weeks.In contrast,BCL6 expression continued to rise.更多还原