【作者】 刘光辉；

【导师】 曾溢滔；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2020， 博士

【摘要】 在原肠运动过程中,原条的位置上出现一类新的细胞将上胚层和下胚层分隔开,并且形成新的胚层,这层新的细胞称为中胚层细胞。中胚层细胞将来分化为血液、骨骼、肌肉、肾脏、结缔组织、真皮、泌尿生殖系统等重要的组织和器官。在转录组层面对早期中胚层细胞进行高通量数据分析,能够系统而客观地揭开早期中胚层细胞特性神秘面纱。本研究选择小鼠原肠后期的胚胎为研究对象,观察胚胎的形态特征,利用激光捕获显微切割技术获得外胚层、中胚层和内胚层,随后用微量细胞RNA-seq技术获得转录组信息,通过生物信息学分析筛选得到了50个在中胚层细胞表达的特征性分子,其中Cdh2、Cdh11、Jag1、Pcdh7和Pdgfra通过q PCR验证了其在中胚层细胞的表达水平显著高于外胚层和内胚层中的表达水平,通过免疫组化方法验证了Fn1、N-cad和T在中胚层细胞中特异性表达。此外,通过IPA分析构建了中胚层特征性分子之间的调控网络。这些分子表达的蛋白位于细胞内的不同部位,且彼此之间有直接或间接的关系,共同在细胞中组成复杂的调控网络系统。生血的发生是中胚层形成→成血管内皮细胞形成→造血形成的过程。血液系统是维持正常的生命活动,其形成过程受到复杂而又精细的细胞分子调控。血液形成的过程经历了早期中胚层细胞→卵黄囊原始生血→AGM(主动脉-性腺-中肾)原始生血和定向生血→胎肝定向生血→骨髓定向造血的复杂过程。其中,胎肝是胚胎发育期造血的关键位置,造血干细胞在胎肝中大量扩增,并向骨髓内造血变迁,能够高效分化为髓系细胞和淋巴细胞。因此,胎肝是研究早期中胚层细胞造血发生的一个很好的器官模型。研究早期胚胎中胚层特异的细胞表面标记分子或者调控因子对于胚胎造血形成的影响,将有助于为提高胚胎干细胞或诱导多能干细胞体外向造血细胞分化的效率提供重要的理论依据。为此,本研究选取所筛选出的中胚层相关基因Pcdh7为研究对象,以胎肝血细胞为研究对象,以此来探究胎肝中Pcdh7~+血细胞是否具是一类具有造血功能的亚群细胞。研究表明,在E13.5-E16.5胎肝发育过程中,Pcdh7~+细胞在有核细胞中的比例逐渐升高(6.4%至19.0%)。对E13.5胎肝Pcdh7~+细胞群进行细胞组分分析,发现Pcdh7~+细胞群含有多种高增殖能力的造血祖细胞,且细胞群大多表达AA4.1、CD117、CD144、Sca-1和CD45等造血细胞相关的标志物,胎肝中MNCs-Pcdh7~+细胞比MNCs-Pcdh7~-细胞含有更多比例的造血细胞。另外,通过体内造血重建实验证实了Pcdh7~+细胞群具有造血重建能力,此亚群细胞含有长期造血能力的造血干细胞。本课题筛选获得了原肠后期胚胎的中胚层的特征性分子,并且对部分特征性分子进行了验证。随后在中胚层细胞候选基因对于中胚层组织形成的相关性研究中,选择Pcdh7~+胎肝血细胞为研究对象,研究分析其生血潜能。结果表明Pcdh7~+细胞亚群含有具有完全造血重建能力的造血干细胞。以上结果暗示早期中胚层的特征性分子可以提前指示下游的某些造血细胞的出现。因此,本研究为早期中胚层细胞的捕获以及为进一步深入研究中胚层向造血细胞分化的机制提供理论参考依据。更多还原

【Abstract】 The mesoderm derives from a new layer between epiblast and hypoblast during gastrulation,and the vertebrate mesoderm can differentiate into various tissues including blood,skeleton,muscles,kidney,connective tissue,dermis,and urogenital system.The high throughput analysis of the transcriptome of early embryonic mesoderm can be used to systematically and objectively clarify the characteristics of mesoderm.The late-gastrula mouse embryo was chosen to study the mesoderm markers.The morphology of late-gastrula was identified before three germ layers cells were obtained by laser capture microdissection.The transcriptome profiles of three germ layers cells were obtained by micro cells RNA-seq.We found 50 specific markers expressed in mesoderm cells after transcriptome analysis by bioinformatic methods.Cdh2,Cdh11,Jag1,Pcdh7 and Pdgfra were significantly expressed in mesoderm more than the other two germ layers at m RNA level identified by q PCR.Fn1,N-cad and T were specifically expressed in mesoderm at protein level identified by immunohistochemical staining.Moreover,regulatory networks among mesoderm markers were constructed by IPA.The proteins expressed by these markers are located in different positions within the cell and connect each other through direct or indirect relationship to support a complicated regulatory network.Hemogenesis is a progress consisting of mesoderm formation,hemangioblast formation and blood formation.Blood is one of the most important constitutions for maintaining life activity.Blood derivation process is regulated by complex and sophisticated molecular events.Hematogenesis is a complicated progress involving mesoderm formation in intra-embryo,primitive hematogenesis in yolk sack,primitive and definitive hematogenesis in AGM region,definitive hematogenesis in fetal liver and definitive hematogenesis in bone marrow.Fetal liver is one of key hematopoietic sites and provide a microenvironment for hematopoietic stem cell to expand and differentiate into erythroid cells,myeloid cells and lymphoid cells.Therefore,fetal liver is a versatile organic model for hematopoietic research in early embryonic development.The characteristic marker genes of mesoderm in gastrula is not clear.Whether the specific molecules expressed in mesoderm could indicate appearance of some subsequent hematopoietic cells in hematopoietic sites.The answers to these questions are very valuable to figure out embryonic stem cells or inducible pluripotent stem cells differentiated into hematopoietic cells in vitro.Furthermore,Pcdh7 as a mesoderm marker gene and fetal liver as a hematopoietic organ were chosen to study whether Pcdh7~+cells in fetal liver have the hematopoietic ability.The percentage of Pcdh7~+cells in fetal liver nucleated cells was from 6.4%to 19.0%during E13.5to E16.5 fetal liver development.The composition of Pcdh7~+cells was analyzed thereafter.A variety of hematopoietic progenitors with highly proliferative potential were involved in Pcdh7~+cells and AA4.1,CD117,CD144,Sca-1 and CD45 as hematopoietic markers were mainly expressed in Pcdh7~+cells.Moreover,the number of hematopoietic cells within Pcdh7~+cells was more than Pcdh7~-cells in fetal liver.The blood reconstitution potential of Pcdh7~+cells was identified in vivo,moreover,long-term hematopoietic stem cells with self-renewal ability were also identified within Pcdh7~+cells.In this study,we obtained mesoderm markers of late-gastrula mouse embryo and identified some of mesoderm markers by experiments.In the study of relevance between mesoderm marker genes and mesoderm derived tissues,Pcdh7~+cells in fetal liver were chosen to indentify its hematopoietic potential.The results indicated that hematopoietic stem cells with total blood reconstitution ability were included in Pcdh7~+cells.Above all,the results indicate that mesoderm markers may indicate the appearance of some kinds of hematopoietic cells thereafter.The results can pave the way for mesoderm isolation and research of mechanism of differentiation from mesoderm to hematopoietic cells.更多还原