红系造血岛巨噬细胞的鉴定及生物学特征研究

中文摘要
中文摘要
研究背景和目的：
红系造血岛(Erythroblastic island, EBI)由一个中央巨噬细胞及围绕其周围的不同分化阶段的红细胞组成，是第一个被发现的造血龛。

EBI巨噬细胞对红系发育具有重要调控作用，是红系发育领域的热点和难点。

多年来研究者发现了多种可能的EBI巨噬细胞表面分子标记，EBI巨噬细胞调控红系发育的理论不断得到完善，然而由于始终未能发现鉴定和分离高纯度EBI巨噬细胞的方法，EBI 巨噬细胞本身功能及其支持红系发育机理的研究受到了很大的阻碍。

鉴于促红细胞生成素(Erythropoietin, EPO) 对红细胞生成是必需的，EPO受体(Erythropoietin Receptor, EPOR) 亦在多种非红系细胞中表达。

因此，我们推测EBI巨噬细胞表达EPOR，这样EPO就可以同时作用于红系细胞以及EBI巨噬细胞从而保证高效的红细胞生成。

为了验证这一假说，本课题拟结合Epor-eGFPcre小鼠模型、人胎肝以及体外共培养体系，综合运用流式细胞术、Imagestream、RNA-seq、单细胞测序(Single cell RNA-sequence, Sc-RNA-seq) 等方法对EBI巨噬细胞进行详细系统地研究，构建EBI巨噬细胞鉴定和分离体系，明确其生物学特征，为深入了解EBI巨噬细胞支持红系发育的作用机理提供新的理论，也为未来研究EBI巨噬细胞在地中海贫血(β-thalassemia)、真性红细胞增多症(Polycythemia vera, PV) 和骨髓增生异常综合征(Myelodysplastic syndrome, MDS) 等血液系统疾病中的作用提供重要的依据。

研究方法
(1)EBI巨噬细胞的鉴定、分离和免疫表型分析：利用流式细胞术结合细胞计数证明并不是所有的F4/80+Vcam1+CD169+巨噬细胞都是EBI巨噬细胞；在Epor-eGFPcre小鼠骨髓和胎肝以及人胎肝中，利用流式细胞术、流式细胞分选术和Imagestream方法鉴定并分离小鼠骨髓和胎肝以及人胎肝EBI巨噬细胞，并根据已知的对红系发育起重要作用的表面标记物分析其免疫表型。

(2)EPO促进EBI形成的研究：小鼠进行EPO注射后，利用流式细胞术、
中文摘要
细胞计数、Imagestream对小鼠骨髓和脾脏中有核红细胞数目、EBI巨噬细胞数目、EBI数目以及小鼠骨髓EBI巨噬细胞粘附分子CD163、CD169和Vcam1进行检测；检测人CD34+造血干细胞来源的巨噬细胞CD163、CD169和EPOR的表达，EPOR的表达同时利用EPO刺激后的信号转导进行验证；晚期红系细胞与人EBI巨噬细胞体外共培养检测EPO对人EBI形成的影响。

(3)EBI巨噬细胞转录组学分析：利用分离小鼠骨髓EBI巨噬细胞的方法，分选出高纯度的小鼠骨髓EBI巨噬细胞，进行RNA-seq检测，并应用生物信息学软件进行转录组学分析；用同样的方法对人胎肝EBI巨噬细胞进行RNA-seq 测序和转录组学分析；对RNA-seq检测发现的特异高表达分子进行qRT-PCR和流式细胞术或者Western Blot检测在mRNA和蛋白水平分别进行验证。

(4)人胎肝EBI巨噬细胞异质性研究：利用流式降维分析法、Cytospins 以及Imagestream分析人胎肝不同类型的EBI；分选出人胎肝EBI巨噬细胞进行10X Genomic单细胞测序，利用生物信息学软件进行分析，阐释不同功能的EBI巨噬细胞亚群。

结果
EBI巨噬细胞的鉴定、分离和免疫表型分析
(1)定量结果显示小鼠骨髓中F4/80+Vcam1+CD169+巨噬细胞数：有核红细胞数为1:2.6，而文献报道一个EBI巨噬细胞其周围围绕有核红细胞数目为5-30个；因此，并不是所有的F4/80+Vcam1+CD169+巨噬细胞都是EBI巨噬细胞；小鼠骨髓中约5%的F4/80+巨噬细胞是Epor-eGFP+，胎肝中约31%的F4/80+巨噬细胞是Epor-eGFP+；分选小鼠骨髓和胎肝F4/80+Epor-eGFP+和F4/80+Epor-eGFP-巨噬细胞发现他们形态差异很大，提示他们属于不同的巨噬细胞亚群。

(2)Imagestream检测发现小鼠骨髓和胎肝中超过90%的EBI是由F4/80+Epor-eGFP+巨噬细胞形成的，其中小鼠骨髓中EBI巨噬细胞周围围绕3个、4个和5个及以上有核红细胞的比例分别是约20%、26%和54%；而小鼠胎肝中EBI巨噬细胞周围围绕3个、4个和5个及以上有核红细胞的比例分别是约10%、20%和70%；Imagestream结果显示在小鼠骨髓和胎肝中超过90%的吞噬红细胞核的巨噬细胞是F4/80+Epor-eGFP+；Imagestream结果显示小鼠骨髓中超过90%
中文摘要
的EBI巨噬细胞表达Vcam1、CD169和ER-HR3，约35%的EBI巨噬细胞表达CD163，约52%表达Ly6C以及约32%表达CD11b；之外，Ly6G之前被认为是EBI巨噬细胞的一个分子标记，在所有EBI巨噬细胞中均不表达。

(3)流式细胞术检测结果显示小鼠骨髓中所有的F4/80+Epor-eGFP+巨噬细胞和F4/80+Epor-eGFP-巨噬细胞均表达CD45；大部分F4/80+Epor-eGFP+巨噬细胞是CD11b medi/-，而68% 的F4/80+Epor-eGFP-巨噬细胞表达CD11b；约90%的F4/80+Epor-eGFP+巨噬细胞表达ER-HR3，而仅约52%的F4/80+Epor-eGFP-巨噬细胞表达ER-HR3；虽然大部分F4/80+Epor-eGFP+巨噬细胞和F4/80+Epor-eGFP-巨噬细胞均表达CD169和Vcam1，但是对比F4/80+Epor-eGFP-巨噬细胞，F4/80+Epor-eGFP+巨噬细胞具有较高水平的CD169和Vcam1的表达；F4/80+Epor-eGFP+巨噬细胞中约35%表达CD163，而F4/80+Epor-eGFP-巨噬细胞均不表达CD163；F4/80+Epor-eGFP+巨噬细胞中约69%表达Ly6C，而F4/80+Epor-eGFP-巨噬细胞中约57%的细胞表达Ly6C；这些结果提示F4/80+Epor-eGFP+和F4/80+Epor-eGFP-巨噬细胞具有明显的免疫表型差异。

(4)流式细胞术检测结果显示在人胎肝CD163+巨噬细胞中约27%表达EPOR，Imagestream结果显示超过90%的人胎肝EBI是由CD163+EPOR+巨噬细胞形成的；分选的CD163+EPOR+巨噬细胞和CD163+EPOR-巨噬细胞形态差异很大，且在CD163+EPOR+巨噬细胞中可观察到吞噬细胞核和衰老红细胞的现象。

EPO促进EBI形成的研究
(1)分选的小鼠骨髓F4/80+Epor-eGFP+巨噬细胞具有接受EPO刺激后信号转导反应，而F4/80+Epor-eGFP-巨噬细胞对EPO无反应；EPO注射后导致小鼠骨髓有核红细胞、EBI巨噬细胞以及EBI数明显增加；EBI增多的原因至少部分是由于EPO注射后EBI巨噬细胞Vcam1表达升高引起的；EPO注射后脾脏代偿性造血的EBI也是由F4/80+Epor-eGFP+巨噬细胞形成的，脾脏有核红细胞、EBI巨噬细胞以及EBI数目亦明显增加。

(2)人脐带血CD34+造血干细胞来源的巨噬细胞表达CD163、CD169和EPOR；EPO刺激后STA T5和ATK磷酸化明显升高进一步证明了EPOR的表达；体外岛屿形成实验显示EPO可以促进人EBI的形成。

中文摘要
小鼠和人EBI巨噬细胞的转录组学分析
(1)小鼠骨髓巨噬细胞RNA-seq结果提示F4/80+Epor-eGFP+巨噬细胞具有特殊的支持红系发育的功能如红系发育、受体介导的内吞、凋亡细胞清除、铁稳态等，而F4/80+Epor-eGFP-巨噬细胞则主要与免疫反应和炎症反应等相关。

重要的是，已知的与红系发育息息相关的分子如生长因子Igf1、Il18等，粘附分子Vcam1、CD169和CD163，铁代谢分子以及红细胞核吞噬和消化相关分子Mertk、Timd4、Dnase2α、Trf、Homox1、Slc40a1等均在F4/80+Epor-eGFP+巨噬细胞中高表达。

除此之外，四个转录因子Klf1、Spic、Nr1h3和Maf 选择性地在F4/80+Epor-eGFP+巨噬细胞中表达，提示其在EBI巨噬细胞中的选择性调控作用。

(2)人胎肝巨噬细胞RNA-seq结果提示人胎肝EBI巨噬细胞上调的信号通路主要与脂类代谢、受体介导的内吞、细胞粘附、细胞外基质重塑等功能相关，而CD163+EPOR-巨噬细胞主要和免疫反应、炎症反应、趋化等功能相关；CD163+EPOR+巨噬细胞高表达促进早期红细胞增殖的细胞因子IGF2、CXCL12等，与红细胞粘附的分子VCAM1、CD163、CD169、EMP等，吞噬和铁代谢相关分子如MERTK、AXL、TIMD4、HMOX1、SLC40A1、TRF等；除此之外，转录因子SPIC、NR1H3、PPARG、KLF1、TLX1、EPAS1和ETV5选择性地表达于CD163+EPOR+巨噬细胞，提示其对EBI巨噬细胞的选择性调控作用。

人胎肝EBI巨噬细胞异质性研究
(1)Cytospins和Imagestream结果显示人胎肝存在不同类型的EBI，如周围主要围绕早期红细胞的，周围主要围绕晚期红细胞的，吞噬红细胞脱出的细胞核的等；流式降维分析结果显示人胎肝EBI巨噬细胞存在明显异质性。

(2)ScRNA-seq分析结果显示人胎肝EBI巨噬细胞存在不同的功能亚群，主要有四群：一群是分泌细胞因子支持早期红系细胞增殖的；一群是周围围绕不同期别红细胞，监视红系发育进程清除异常红细胞的；一群是为晚期红细胞成熟供铁的；一群主要起到消化红细胞核。

结论：
(1)在小鼠骨髓和胎肝以及人胎肝中EBI巨噬细胞表达EPOR，且EPO可以通过调控其功能促进EBI形成。

中文摘要
(2)EBI巨噬细胞通过分泌生长因子、直接与红细胞粘附、为晚期红细胞合成血红蛋白提供铁以及吞噬红细胞脱出的细胞核等方式支持红系发育。

(3) EBI巨噬细胞存在异质性，在人胎肝中存在四个不同功能的EBI巨噬细胞亚群。

关键词：红系造血岛；巨噬细胞；红系发育；促红细胞生成素受体；异质性；单细胞测序。

Abstract
Abstract
Background and purpose
The erythroblastic island, composed of a central macrophage and surrounding erythroid cells, was the first hematopoietic niche discovered. The regulation of EBI macrophages during erythropoiesis is a research hotspot and difficult issue in the field of erythropoiesis. For many years, researchers have found a variety of possible surface markers of EBI macrophages. However, due to the lack of isolation of EBI macrophages, the identity of EBI macrophages per se and the regulation of erythropoiesis has thus far remained elusive. Given that EPO is essential for erythropoiesis and that Epor is expressed in numerous non-erythroid cells, we hypothesized that EBI macrophages express Epor so that EPO can act on both erythroid cells and EBI macrophages simultaneously to ensure efficient erythropoiesis. To test this notion, we employed Epor-eGFPcre knock in mouse model, human fetal liver (FL) and in vitro co-culture system to study the EBI macrophages systematically. Flow cytometry, Imagestream, RNA-seq, Single cell RNA-sequence (Sc-RNA-seq) and other methods were used to conduct the identification and isolation of EBI macrophages system to clarify the biological characteristics of EBI macrophages. The project will provide important resources for future studies of EBI macrophage function during normal as well as disordered erythropoiesis in hematologic diseases such as β-thalassemia, PV, MDS and so on.
Research methods
(1)Identification, isolation and immunophenotypically analysis of EBI macrophages: To determine whether all the F4/80+Vcam1+CD169+ macrophages are EBI macrophages, we quantified the numbers of erythroblasts and F4/80+Vcam1+CD169+macrophages in mouse BM. Then, we isolated EBI macrophages from Epor-eGFPcre mouse BM, FL and Huam FL using flowcytometry,
Abstract
cell sorting and Imagestream. Immunophenotypic was analyzed based on known surface molecules, which were reported to support erytghropoiesis.
(2)The study of EPO on EBI formation: Then, we calculated the numbers of EBI, EBI macrophage, and erythroblast using flow cytometry, imagestream and cell counting in both mouse BM and SP after EPO injection; The expression of adhension mulecules CD163, CD169 and Vcam1 in mouse BM EBI macrophages was determined using flow cytometry after EPO injection at the same time; The expression of CD163, CD169 and EPOR were checked in human CD34+ cells derived macrophages by flow cytometry and the expression of EPOR were confirmed by EPO stimulation experiment; EBI forming assay was performed using late stage erythroblasts and human CD34+ cell-derived macrophages to test the hypothesis EPO plays a role in EBI formation by acting on EPO-responsive EBI macrophages.
(3)Transcriptome analysis of EBI macrophages: RNA-seq was performed on both mouse BM EBI macropahges and human FL EBI macrophages; Bioinformatic softwares were used to analyze the data to develop a comprehensive characterization of the EBI macrophages at the molecular level in both mouse and man. qRT-PCR and Flowcytometry or Western Blot were used to confirm both mRNA and protein levels of specific-high expression molecules from RNA-seq.
(4)The heterogeneity of human FL EBI macrophages: The different types of EBI were confirmed using flow dimensionality reduction analysis, cytospins and imagestream in human FL. 10X genomic Sc-RNA-seq was performend for both sorted CD163+EPOR+ and CD163+EPOR- macrophages. The data was analyzed using bioinformatic software to interpret different functional subpopulations of EBI macrophages.
Results
Identification, isolation and immunophenotypically analysis of EBI macrophages
(1)Calculation of the cell numbers revealed that there were ~4 million F4/80+Vcam1+CD169+macrophages and ~10.5 million erythroblasts in mouse BM cells from two femurs and two tibias, giving a ratio of F4/80+Vcam1+CD169+ versus
Abstract
erythroblasts about 1:2.6, strongly suggesting that it is unlikely that all the F4/80+Vcam1+CD169+macrophages are EBI macrophages. About 5% and 31% of F4/80+macrophages express Epor-eGFP in mouse BM and FL, respectively. The sorted F4/80+Epor-eGFP+ macrophages and F4/80+Epor-eGFP-macrophages are morphologically different in both mouse BM and FL, further demonstrating that they are indeed distinct macrophage populations.
(2) Imagestream results revealed that more than 90% EBIs were formed by the F4/80+Epor-eGFP+macrophages in both mouse BM and FL. Further analyses showed that the percentages of EBIs with 3, 4, and 5 or more erythroid cells were ~20%, ~26% and ~54%, respectively in mouse BM. And the percentages of EBIs with 3, 4, and 5 or more erythroid cells were ~10%, ~20% and ~70%, respectively in mouse FL. Imagestream results also revealed that more than 90% of macrophages phagocytizing nuclei were F4/80+Epor-eGFP+ macrophages in both BM and FL. Finally, our results demonstrated that Vcam1, CD169, and ER-HR3 were expressed on more than 90% of F4/80+Epor-eGFP+EBI macrophages, while CD163, Ly6C and CD11b were expressed on about ~35%, ~52%, ~32% of the F4/80+Epor-eGFP+ EBI macrophages, respectively. Additionally, Ly6G, previously implicated as an EBI macrophage marker, was not expressed on the F4/80+Epor-eGFP+EBI macrophages.
(3) Flowcytometry results showed that all F4/80+Epor-eGFP+macrophages were CD45+, almost all of them expressed Vcam1 and CD169, a proportion of them expressed CD163 (~35%), Ly6C (~69%) or ER-HR3(~90%), and some of them express moderate levels of CD11b. Our results also showed that F4/80+EporeGFP-macrophages were CD45+. In contrast, although almost all F4/80+Epor-eGFP-macrophages also expressed Vcam1 and CD169, but expression levels of Vcam1 and CD169 on the F4/80+Epor-eGFP-macrophages were low. Moreover, the F4/80+Epor-eGFP-macrophages did not express CD163, and a proportion of them expressed Ly6C (~57%), ER-HR3(~52%) and CD11b (~68%). These results indicate that mouse BM F4/80+Epor-eGFP+macrophages are immunophenotypically distinct from F4/80+Epor-eGFP- macrophages.
(4) In human FL, within the CD163+macrophages, ~27% were EPOR+. Cytospin images showed that CD163+EPOR+ macrophages were morphologically different
Abstract
from CD163+EPOR- macrophages; Imagestream revealed that human FL native EBIs were also formed by CD163+EPOR+ macrophages.
The study of EPO on EBI formation
(1) The expression of Epor in mouse BM F4/80+Epor-eGFP+ EBI macrophages was further confirmed by the finding that EPO treatment led to phosphorylation of STAT5. EPO injection lead to an ~3 folds increase in the number of EBIs with a concomitant increase in EBIs with 5 or more erythroid cells. The increase in EBIs was accompanied by an increase in both erythroblasts and F4/80+Epor-eGFP+ macrophages, indicating the function of these Epor-eGFP+ EBI macrophages in EPO induced stress erythropoiesis. EPO injection resulted in increased surface expression of Vcam1 but not CD169 on the F4/80+Epor-eGFP+macrophages. Although EPO injection did not increase the surface expression of CD163, it increased the percentage of CD163in F4/80+Epor-eGFP+ macrophages. Importantly, stress splenic EBIs were also formed by the F4/80+Epor-eGFP+macrophages and the numbers of erythroblast, EBI macrophage and EBI were also significantly increased.
(2)EPOR, along with CD163, and CD169 were expressed on CD34+ cell-derived macrophages. EPOR expression was further confirmed by the finding that EPO treatment led to phosphorylation of STAT5 and AKT, downstream targets of EPO/EPOR mediated signal transduction. EPO plays a significant role in EBI formation by acting on EPO-responsive EBI macrophages in vitro.
Transcriptome analysis of mouse and human EBI macrophages
(1) Gene Ontology (GO) analysis of the differentially expressed genes revealed that the top upregulated pathways in F4/80+Epor-eGFP+macrophages included erythrocyte development, receptor-mediated endocytosis, apoptotic cell clearance and cellular iron ion homeostasis, In contrast, the top upregulated pathways in F4/80+Epor-eGFP-macrophages were mostly related to immune and inflammatory response. Specifically, the expression levels of genes encoding proteins known to be important for EBI macrophage function in supporting erythropoiesis were significantly higher in F4/80+Epor-eGFP+macrophages than in F4/80+Epor-eGFP-macrophages. These included growth factor Igf1, Il18 and so on, adhesion molecules Vcam1, CD169 and CD163, iron metabolism molecules, phagocytosis and digestion
Abstract
related molecules Mertk, Timd4,Dnase2a, Trf, Homox1, Slc40a1and so on. Additionally, four transcriptional factors (TFs) Klf1, Spic, Nr1h3and Maf are expressed in the F4/80+Epor-eGFP+macrophages but not in the F4/80+Epor-eGFP-, suggesting their selective roles in transcriptional regulation of the EBI macrophages.
(2) Gene Ontology (GO) analysis of the differentially expressed genes revealed that the top upregulated pathways in CD163+EPOR+macrophages included lipoprotein metabolic process, receptor-mediated endocytosis, cell adhesion and extracellular matrix orgnization. In contrast, the top upregulated pathways in CD163+ EPOR- macrophages were mostly related to inflammatory response, immune response and chemotaxis. Specifically, the expression levels of genes encoding proteins known to be important for EBI macrophage function in supporting erythropoiesis were significantly higher in CD163+EPOR+macrophages than in CD163+EPOR- macrophages. These included growth factors IGF2 and CXCL12, adhesion molecules CD163, CD169, VCAM1, and EMP, iron metabolism molecules, phagocytosis and digestion related molecules HMOX1, SLC40A1, TIMD4, AXL, MERTK, TRF and so on. Additionally, seven TFs SPIC, NR1H3, PPARG, KLF1, TLX1, EPAS1 and ETV5 are expressed in the CD163+EPOR+ macrophages but not CD163+EPOR- macrophages, suggesting their selective roles in transcriptional regulation of the EBI macrophages. These findings strongly suggest that the CD163+EPOR+ macrophages in huam FL have evolved a specialized function in supporting erythropoiesis.
The heterogeneity of human FL EBI macrophages
(1)Cytospins and Imagestream results showed that different types of EBI existed in human FL, and the results of flow dimensionality reduction analysis showed that EBI macrophages were significantly heterogeneous.
(2)The ScRNA-seq results showed that there were four different functional subpopulations of EBI macrophage in human FL. One cluster mainly secreted growth factors to support the proliferation of early stage of erythroblasts; One cluster surrounded by different stages of erythroblasts, which surveillanced the process of erythropoiesis and cleard the abnormal erythroid cells; One cluster was considered to support the maturation of late stage of erythroblasts and one cluster was mainly
Abstract
correlated with digesting the erythroblast nucleus.
Conclusions
(1)EBI macrophages express EPOR in both mouse BM, FL and human FL, which response to EPO and then promote EBI formation.
(2)EBI macrophages promote erythropoiesis by secreting growth factors, directly adhering to erythroblasts, providing iron for late stages of erythroblasts, and phagocytosising pyrenocyte.
(3) EBI macrophages are heterogeneous and there are four different functional subpopulations of EBI macrophage in human fetal liver.
Key words:Erythroblastic island; macrophages; erythropoiesis; EPOR; single-cell RNA-seq.
XI
目录
目录
缩略词表 (I)
第一章概述 (1)
1.1文献综述 (1)
前言 (1)
（一）红系造血岛的组成、类型及潜在成熟机制 (2)
（二）红系造血岛在小鼠、人和大鼠中调控红系发育的研究 (3)
（三）巨噬细胞-红细胞相互作用和EBI巨噬细胞的免疫表型特征。

(4)
（四）EBI巨噬细胞和铁代谢 (7)
（五）巨噬细胞分泌生长因子影响红系发育 (7)
（六）展望 (8)
1.2研究意义 (9)
参考文献 (11)
第二章EBI巨噬细胞的鉴定及转录组学分析 (19)
2.1前言 (19)
2.1.1 红系发育及相关疾病 (19)
2.1.2 红细胞自身调控机制研究进展 (19)
2.1.3 巨噬细胞对红系发育的调控作用 (20)
2.1.4 巨噬细胞与红细胞之间的粘附分子 (20)
2.1.5 本研究的目的和意义 (21)
2.2材料和方法 (22)
2.2.1 实验材料 (22)
2.2.2 实验方法 (24)
2.3实验结果 (35)
一、EBI巨噬细胞的鉴定、分离和免疫表型分析 (35)
2.3.1 并不是所有的F4/80+Vcam1+CD169+巨噬细胞都是EBI巨噬细胞 (35)
目录
2.3.2 在小鼠骨髓和胎肝F4/80+巨噬细胞中存在表达Epor-eGFP的亚群 (37)
2.3.3 小鼠骨髓和胎肝F4/80+Epor-eGFP+和F4/80+Epor-eGFP-巨噬细胞形态差异很大
(38)
2.3.4 小鼠骨髓EBIs是由F4/80+Epor-eGFP+巨噬细胞形成的 (39)
2.3.5 小鼠胎肝EBIs是由F4/80+Epor-eGFP+巨噬细胞形成的 (41)
2.3.6 小鼠骨髓和胎肝红细胞核由F4/80+Epor-eGFP+巨噬细胞所吞噬 (42)
2.3.7 小鼠骨髓F4/80+Epor-eGFP+和F4/80+Epor-eGFP-巨噬细胞表达不同的单核/巨噬
细胞表面标记 (43)
2.3.8 小鼠骨髓EBI巨噬细胞表达不同的表面标记 (43)
2.3.9 人EPOR流式抗体可以识别EPOR的表达 (45)
2.3.10 人胎肝EBI巨噬细胞表达EPOR (46)
2.3.11 人胎肝红细胞核由CD163+EPOR+巨噬细胞吞噬 (47)
二、EPO对小鼠和人EBI形成的影响 (48)
2.3.12 小鼠骨髓EBI巨噬细胞表达功能性Epor (48)
2.3.13 EPO增加小鼠骨髓EBI的形成 (49)
2.3.14 EPO增加小鼠骨髓EBI巨噬细胞Vcam1的表达 (49)
2.3.15 在EPO应激时小鼠脾脏EBI由F4/80+Epor-eGFP+巨噬细胞形成 (50)
2.3.16 EPO增加人EBI的形成 (51)
2.3.17 EPO不增加人EBI巨噬细胞CD163、CD169以及MAEA的表达 (52)
三、小鼠骨髓和人胎肝EBI巨噬细胞的转录组学分析 (53)
2.3.18 小鼠骨髓Epor-eGFP+和Epor-eGFP-巨噬细胞差异表达分析 (53)
2.3.19 小鼠骨髓Epor-eGFP+巨噬细胞高表达支持红系发育的重要分子 (54)
2.3.20 mRNA水平验证小鼠骨髓Epor-eGFP+巨噬细胞特异高表达基因 (55)
2.3.21 蛋白水平验证小鼠骨髓Epor-eGFP+巨噬细胞特异高表达分子 (56)
2.3.22 人胎肝CD163+EPOR+和CD163+EPOR-巨噬细胞差异表达分析 (57)
2.3.23 人胎肝CD163+EPOR+巨噬细胞高表达支持红系发育的重要分子 (58)
2.3.24 人胎肝CD163+EPOR+巨噬细胞重要表面标记的验证 (59)
2.4讨论 (60)
第三章人胎肝EBI巨噬细胞功能异质性研究 (66)
目录
3.1前言 (66)
3.1.1 EBI巨噬细胞异质性研究进展 (66)
3.1.2 单细胞测序在EBI巨噬细胞异质性检测中应用前景 (66)
3.1.3 本研究的目的及意义 (67)
3.2材料和方法 (67)
3.2.1 利用Single cell-RNA-seq技术分析EBI巨噬细胞的异质性 (68)
3.3实验结果 (68)
3.3.1 人胎肝EBI巨噬细胞具有免疫表型异质性 (68)
3.3.2 Imagestream 和Cytospins 显示人胎肝不同类型EBI的存在 (69)
3.3.3 分选人胎肝EBI巨噬细胞进行单细胞测序流程图 (70)
3.3.4 人胎肝CD163+EPOR-和CD163+EPOR+巨噬细胞分群结果 (71)
3.3.5 人胎肝CD163+EPOR-巨噬细胞以及红细胞亚群标志性基因 (72)
3.3.6 人胎肝CD163+EPOR+巨噬细胞亚群标志性基因 (75)
3.3.7 人胎肝CD163+EPOR+和CD163+EPOR-巨噬细胞标志性信号通路 (77)
3.4讨论 (78)
第四章全文总结及进一步研究意义 (83)
参考文献 (85)
个人简历、在学期间发表学术论文及研究成果 (94)
致谢 (96)
缩略词表
缩略词表
缩略词英文全称中文全称
CXCL12 DNASE1L3 Epor C-X-C motif chemokine ligand 12
Deoxyribonuclease 1 like 3
Erythropoietic receptor
C-X-C基序趋化因子配体12
脱氧核糖核酸酶1类似物3
促红细胞生成素受体
EMP Erythroblast macrophage protein 红细胞巨噬细胞蛋白
EBI HMOX1 HIF-2 αIgf1
Il18 IGFBP4 ICAM4 KLF1 MARCO MDS NR1H3 PPARG PV RNA-seq RNASE1 Erythroblastic island
Heme oxygenase 1
hypoxia-inducible factor-2 α
Insulin growth factor 1
Interleukin 18
Insulin growth factor binding protein 4
Intercellular adhesion molecule 4
Krüppel-like factor 1
Macrophage receptor with collagenous structure
Myelodysplastic syndromes
Nuclear receptor subfamily 1 group H member 3
Peroxisome proliferator activated receptor gamma
polycythemia vera
RNA-sequences
Ribonuclease A family member 1
红细胞岛屿
血红素加氧酶1
乏氧诱导因子2 α
胰岛素样生长因子1
白细胞介素18
胰岛素样生长因子结合蛋白 4
细胞间粘附分子4
Krüppel样因子1
具有胶原结果的巨噬细胞受体
骨髓增生异常综合征
核受体亚家族1组H成员3
过氧化物酶体增殖物激活受体γ
真性红细胞增多症
RNA测序
核糖核酸酶A家族成员1
Sc-RNA-seq SIGLEC1 SLC40A1 Single cell RNA-seq
Sialic acid binding Ig like lectin 1
Solute carrier family 40 member 1
单细胞RNA测序
唾液酸结合Ig样凝集素1
膜铁转运蛋白
TIMD4 T-cell immunoglobulin and mucin domain containing 4 T细胞免疫球蛋白和粘蛋白结构域含有4
Vegfb Vascular endothelial growth factor b 血管内皮生长因子b Vcam1 Vascular cell adhesion molecule 1 血管细胞粘附分子1
第一章概述
第一章概述
1.1 文献综述
前言
红系发育是一个具有阶段特异性的精细分化过程。

红细胞的生成历经由多能造血干细胞向红系祖细胞定向分化以及红系祖细胞继续发育为成熟红细胞等过程。

多能造血干细胞分化为多能髓系祖细胞后，在促红细胞生成素(Erythropoietin, EPO)作用下定向分化为早期红系祖细胞红系爆式集落形成单位(Burst forming unit-erythroid, BFU-E)和晚期红系祖细胞红系克隆形成单位(Colony forming unit-erythroid，CFU-E)，CFU-E继续增殖依次分化成原始红(Proerythroblast, Pro-E)、早幼红(Basophilic erythroblast, Baso-E)、中幼红(Polychromatic erythroblast, Poly-E)、晚幼红(Orthochromatic erythroblast, Ortho-E)和脱核后的网织红细胞(Reticulocyte)，最终形成成熟红细胞(Red blood cells, RBCs)[1–5]。

而红系发育任何一个阶段的异常都将造成红细胞发育不良，导致相关疾病的产生。

红细胞异常性疾病是人类最常见的疾病，全球约有10亿人口患有各种类型的红细胞异常性疾病，包括地中海贫血[6–8]、先天性红细胞生成不良性贫血[9–11]、先天性纯红细胞再生障碍性贫血[12,13]、疟原虫感染引起的贫血[14–16] 以及包括骨髓增生异常综合症(Myelodysplastic Syndrome, MDS) 在内的多种骨髓障碍综合症[17–19]。

然而目前大多红细胞发育异常性疾病的发病机制尚不完全清楚，这严重限制了对该类疾病的有效诊断和治疗，影响患者的健康和生活质量，造成极大的社会负担。

而红系疾病发病机制的研究困境在很大程度上也与红系发育调控机制不明有关。

红系发育过程受到多层次的复杂而精细的调控。

对红系发育自身调控机制的研究过去主要集中在细胞因子、转录因子以及调控珠蛋白基因表达和转换的顺势作用元件等。

其中研究较为清楚的是EPO及其受体(EPO Receptor, EPOR)。

EPO/EPOR 信号通路的激活能够抑制CFU-E 和Pro-E 的凋亡，进而在红系发育进程中起到关键作用，被公认为是红系发育的必要因素[20–24]。

在转录水平，
第一章概述
红细胞发育被多个核心转录因子调节。

其中GATA1 和KLF1 因其在造血过程中的重要作用被认为是最主要的红系发育调控因子[25,26]。

近年来国内外血液学家的研究还发现非编码RNA、MicroRNA、DNA甲基化修饰、组蛋白修饰、剪切因子、泛素化修饰等多种表观遗传调控因子在正常和疾病相关红系发育进程中起到重要的调控作用[3–5,27–35]。

相对于红细胞自身调控因素参与调节红系发育功能的深入了解，目前对红系造血岛(Erythroblastic island，EBI)的了解极为有限。

EBI是体内红细胞生成的特定场所。

无论是在胎儿期的胎肝中还是在成年期的骨髓中，红细胞的生成都离不开EBI的调控与支持作用。

早在1958年法国血液学家Bessis M就根据骨髓透射电镜照片首次发现在红系发育过程中，不同发育阶段的红细胞围绕一个中央巨噬细胞组成特定的细胞发育群落，称之为EBI[36]。

然而关于EBI在红系发育中是否具有调节作用，20年后才由Mohandas等人首次证明，他们发现大鼠过渡输血后骨髓中EBI数目明显减少[37]。

之后科学家通过体外共培养发现巨噬细胞能够促进红系前体细胞的存活和增殖[38–40]。

并且基于多种基因编辑小鼠模型的研究也陆续证明巨噬细胞分化发育异常能够导致红系发育障碍和贫血[41,42]。

近期有研究通过体内实验证明清除巨噬细胞后小鼠在诱导急性溶血时贫血症状恢复明显减慢，从而首次直接证明了巨噬细胞对红系发育具有重要的调控作用[43,44]。

以上的研究表明EBI在红系发育过程中起到至关重要的作用。

在本篇综述中，我们将总结EBI的研究历史及进展，指出尚未被解决的重要科学问题，并展望未来发展趋势。

（一）红系造血岛的组成、类型及潜在成熟机制
60余年前，法国血液学家Marcel Bessis首先描述了EBI，其包括一个中央巨噬细胞和周围围绕的数目不等的红系细胞[36]。

2003年和2016年两项研究利用电子扫描显微镜发现根据岛屿的大小、形态、红细胞不同成熟阶段和相对于血窦的位置不同，有两种不同类型EBI的存在[45,46]。

这两种EBI一种是圆形、较小、周围围绕早期红系细胞且距血窦距离较远的，称之为不成熟EBI；另一种是扁平、较大、周围围绕晚期红系细胞且距血窦距离较近的，称之为成熟EBI。

它们之间强烈的差异表明它们可能在红系发育过程中发挥不同的时空作用。

两种不同类型的EBI及其特征提示我们EBI是不断发育成熟的，成熟后的EBI距离血窦较近，此时红细胞逐渐脱核成熟并离开EBI，成熟后的红细胞通过血窦内皮
第一章概述
细胞释放到血循环中。

此外，不同的研究结果显示，每个EBI周围围绕的成红细胞数量范围为5-30个，这亦提示不同类型EBI的存在[47]。

EBI成熟的机制目前尚不完全清楚。

已知的是在EBI成熟过程中其逐渐向血窦内皮细胞迁移。

最近的研究表明不同来源的内皮细胞促进巨噬细胞分化和极化为M2样巨噬细胞，胎肝中的内皮通过膜蛋白调节组织定居巨噬细胞的迁移、定居和生存[48,49]。

虽然研究显示内皮细胞可能在调节巨噬细胞的成熟和迁移中发挥重要作用，但是仍需要更精确的EBI巨噬细胞成熟、迁移及其对红细胞生成的调控机制。

因此以下基本问题值得进一步研究和明确：1）未成熟的EBI如何分化为成熟的EBI？2）两种不同类型的EBI巨噬细胞调节红细胞生成的机制是什么，有什么区别？3）EBI如何向血窦迁移？
（二）红系造血岛在小鼠、人和大鼠中调控红系发育的研究关于EBI巨噬细胞对红系发育是否具有调控作用首先是由Narla Mohandas 和他的同事证实的，他们的研究结果表明过渡输血的大鼠，其骨髓中EBI的数量明显减少[50]。

之后越来越多的证据显示EBI在小鼠、大鼠和人中对红系发育均具有重要的调控作用。

1）红系造血岛在小鼠红系发育中作用的研究
红细胞生成发生在EBI上，EBI由一个中央巨噬细胞和数个周围围绕的红细胞组成[51]。

关于EBI在小鼠中调控红系发育的研究已有多年的历史，多种基因敲除小鼠模型显示巨噬细胞分化异常导致小鼠贫血症状的发生或者在胚胎期导致死亡[41,42]。

小鼠中清除巨噬细胞以后亦发现小鼠骨髓中有核红细胞数目明显减少，在诱导溶血性贫血时，小鼠贫血症状恢复明显减慢，这直接证明了巨噬细胞对小鼠红系发育具有重要的调控作用[43,44]。

然而与胎肝和骨髓中的巨噬细胞不同，脾脏中的巨噬细胞主要是清除衰老的红细胞，一些数据显示清除小鼠脾脏巨噬细胞后显著影响红细胞的生成[44]，这可能是由于衰老红细胞清除障碍导致的。

最近，美国科学家Robert F. Paulson 教授课题组发现在诱导贫血时单核细胞会被招募到脾脏分化为造血岛巨噬细胞从而促进小鼠贫血症状的恢复[52]。

在巨噬细胞中特异性地敲除EMP的表达亦明显降低小鼠骨髓EBI的形成[62]。

这些研究结果显示在不同的造血器官，在稳态和应激造血时EBI巨噬细胞均对红系发。