【作者】 李芳菲；

【导师】 王亚平；

【作者基本信息】 重庆医科大学 ， 药理学， 2007， 博士

【摘要】 研究背景1998年美国科学家先后从囊胚内细胞团(inner cell mass,ICM)及原始生殖细胞(primordial germ cells, PGCs)中分离得到人ES细胞系(embryonic stem cells)和人EG细胞(embryonic germ cells)。它们在体外具有的无限增殖和多向分化能力使其成为胚胎早期发育研究、细胞治疗和组织工程等领域的重要工具。因此,明确其自我更新和多向分化的调控机制是胚胎干细胞广泛应用的前提。PGCs是指在进入与体细胞有密切接触的生殖腺成为真正的生殖细胞之前,短暂存在于胚胎的双倍体生殖细胞前体。从系统发育学的观点来说,生殖细胞是至关重要的,因为它承载着遗传信息,并使之代代相传,而体细胞则为生殖细胞提供保护和营养。因而,只有了解生殖系从PGCs到成熟配子的发育过程才能洞悉人类胚胎发生的起源。从干细胞生物学的角度而言,原始生殖细胞是成熟配子的前体,将其看作干细胞是无可争议的。hEG细胞培养的困难相对较大。1998年至今仅少数几个实验室宣布获得了hEG细胞,且至今尚未获得可长期培养的hEG细胞,因此优化hEG细胞培养体系必将推动其深入研究。hEG细胞的基本培养基中需添加多种细胞因子以维持其在未分化状态下的增殖,包括白血病抑制因子(Leukemia inhibitory factor,LIF)、干细胞生长因子(Stem cell factor,SCF)、成纤维细胞生长因子(Fibroblast growth factor, FGF)、Forskolin等。同时,hEG细胞的生长还依赖于饲养层细胞的支持。传统培养体系中,饲养层主要采用小鼠成纤维细胞系STO或原代MEF,可能导致培养体系的鼠源性污染。总之,hEG细胞的深入研究和临床应用的前提是建立安全、有效的培养体系。本实验分离人5–9周流产胚胎生殖嵴,以表达LIF基因的人胚肺成纤维细胞(LIF-expressing human embryonic lung fibroblasts, hELF/lif)作为饲养层培养hEGC,并对其生物学特性进行鉴定,旨在为hEG细胞培养体系的建立和优化奠定基础。自2003年以来,已有实验室报道获得ES细胞来源的生殖细胞。2004年底,Science期刊将“体外系统中ES细胞分化产生生殖细胞”的发现列为该年的十大突破进展之一。由胚胎干细胞分化为生殖细胞,除为生殖系统发育提供可研究的模型外,无疑还将为获得成体生殖细胞提供很好的材料,因此具有极大的理论和实际价值。特别的是,EG细胞作为生殖系本身来源的多能干细胞,将其作为向生殖细胞分化的种子细胞较ES细胞更为适合。此外在体内发育过程中,PGCs迁移到生殖嵴,增殖到足够数量后,即触发减数分裂,分化为生殖系干细胞。但是,维持自我更新和触动分化过程中发挥作用的转录调控因子,目前仍不清楚。由于原始生殖细胞缺乏特异性标记,同时受标本来源的限制,使得长期以来对PGCs分化机制的研究较少。hEG细胞来源于生殖系统,本实验将PGCs来源的hEG细胞进行体外“顺势分化”,研究分化过程中多能干细胞标记Oct4、Nanog,生殖系标记Stella、VASA,减数分裂相关标记SCP1、SCP3、GDF9、TEKT1的表达,探讨PGCs增殖分化机制。研究结果一、以转染LIF基因的人胚肺成纤维细胞为饲养层培养人胚胎生殖细胞原代来源的人胚肺成纤维细胞形态典型,增殖旺盛。LIF基因真核表达载体转染后的细胞经G418筛选后得到可传代阳性克隆,经RT-PCR和western-blot鉴定均能稳定表达LIF。分离5-9周胚胎生殖嵴细胞,种植到hELF/lif细胞制成的饲养层上,在培养基中不添加外源性LIF蛋白的条件下,PGCs来源的hEG细胞形成典型的鸟巢状集落,集落呈高度的碱性磷酸酶活性,胚胎特异抗体SSEA-1、SSEA-4、TRA-1-60、TRA-1-81阳性。表达未分化标志Oct-4,Nanog和Rex-1,以及端粒酶活性标记:hTERT。染色体分析培养25天后的细胞为正常人染色体核型。具有hEG细胞的形态学及生物学特性。免疫组化和免疫荧光分析发现:14天EB表达组织特异性标志Desmin和AFP。之后分别对未分化hEG细胞,第7天EB细胞和第14天EB细胞进行的RT-PCR分析发现:随分化程度增加,EB细胞表达三个胚层来源细胞标志,分别为内胚层:α-FP,Pdx-1;中胚层:CD34,enolase;外胚层:Vimentin,NF68,表明hEG细胞具有向三个胚层分化的多向潜能。分别收集在hELF和hELF/lif细胞上生长7天和10天的hEG细胞,进行RT-PCR分析。hEG细胞中能检测到LIFR的表达,但仅在hELF/lif细胞上生长的hEG细胞中检测到LIF信号通路靶基因c-myc的表达,提示在hELF/lif饲养层上生长的hEG细胞中LIF信号通路被激活。二、hEG细胞向生殖细胞分化及其调控机制RT-PCR的方法分析表明:未分化hEG细胞有较高水平的Oct4和Nanog表达,而hEG细胞一旦分化形成为EB(Day3),Nanog表达水平立即显著下降,并随进一步分化下降到检测不到的水平。而Oct4在hEG细胞分化后表达水平下降,但仍保持一定的表达。生殖系标志分子STELLA,PGCs细胞后期(迁移到生殖嵴,减数分裂前)阶段特异性基因VASA从EB形成第7天被检测到,表达水平持续上升,同时,卵母细胞特异性基因GDF9和精原细胞特异性基因TEKT1分别从EB形成第3天,和EB形成第7天开始被检测到。减数分裂标志SCP1在EB中也被检测到。对hEG细胞和EB进行免疫荧光分析发现:未分化hEG细胞胞浆表达Nanog,而EB中表达减数分裂标志分子SCP3的细胞的Nanog表达是缺失的。分别对第7周胚胎和第13周男性及女性胚胎生殖嵴进行检测,结果表明:Nanog在PGCs迁移发育阶段(第7周)大量表达;而PGCs到达生殖嵴后,男性生殖细胞停滞在有丝分裂阶段,女性生殖细胞在进入减数分裂(第13周)后Nanog的表达立即下降到检测不到的水平。因此,Nanog在抑制胚胎生殖细胞分化中可能发挥重要作用。Nanog表达下调是体内触动生殖细胞减数分裂的必要条件,而体外受Oct4转录调控的Nanog表达水平下降可能是促进hEG细胞分化的直接原因。上述结果显示本培养体系中的hEG细胞具有向生殖细胞分化的能力,并具有减数分裂的潜能。结论原始生殖细胞是成体生殖细胞的前体,而成体配子融合又将形成受精卵,从而启动胚胎发育全过程,因此PGCs携带遗传信息起着传承生命的作用,在胚胎发育中具有至关重要的地位,同时又作为体外培养胚胎干细胞的来源之一,具有临床应用的巨大潜力。国内外科研工作者围绕维持PGCs未分化状态及多能性的分子调节机制作了大量研究,但具体机制仍不清楚。进一步探讨hEG细胞自我更新及分化过程中关键因子的功能,具有发育生物学和干细胞治疗学的双重意义。建立无异种动物成分污染的培养系统是胚胎干细胞技术发展的必然趋势。本研究将LIF基因转入hELF细胞中,建立人来源的能分泌LIF活性蛋白的新的饲养层来支持人hEG细胞的体外生长。为优化hEG细胞培养体系奠定基础。在此过程中获得人LIF基因真核表达载体、稳定表达LIF的人胚肺成纤维细胞等材料也可成为多功能细胞因子LIF功能的研究工具。本实验关于hEG细胞向生殖细胞分化过程中分子表达变化的研究,为人类生殖系统发育这一长期困扰生物界的难题提供了线索,也为重要转录因子在胚胎干细胞自我更新中的作用研究提供了证据。在本研究前期建立的以hELF/lif饲养层为基础的hEG细胞培养体系中生长的hEG细胞,分化形成EB,表达生殖细胞和减数分裂标志分子,具有向生殖细胞分化的潜能。更多还原

【Abstract】 BackgroundIn 1998, human embryonic stem cells (ES cells) and embryonic germ cells (EG cells) were successfully established from inner cell mass (ICM) and primordial germ cells (PGCs) respectively. Thus, their unlimited proliferation and multi-lineage differentiation capacity are critical for delineation of early embryo development, cell therapy and tissue engineering, which are largely based on a fully understanding of the regulatory mechanisms regarding selfrenewal and differentiation of embryonic stem cells in vitro.As long term culture of hEG cells is unavailable, optimizing current culture system stands essential for promoting related research. Up to date, various cytokines have been proven required for in vitro undifferentiated growth of hEG cells, including LIF, bFGF, SCF and Forskolin. Moreover, efforts will be made to establish a safe and efficient supportive feed layer to substitute murine fibroblast STO or MEF, ruling out the xenogenic contamination of human embryonic stem cell cultures. Here, hEG cells are inoculated by coculture of cell mixtures from human 5-9 weeks embryo gonads with LIF-integrated human embryonic lung fibroblasts, and a comprehensive characterization is carried out.Prior to enter gonad ridge contacting intimately with somatic cells to become authentic germ cells, PGCs behave transiently as diploid precursors for germ cells. In the opinion of system biology, germ cells are pivotal due to the equipped genetic information to descendents, while somatic cells ensure a variety of protections and nutrients. Therefore, insights of human embryo ontogeny undoubtedly depends on continuous illustrations of development pathway from PGCs to mature gametes. The finding that in vitro generation of germ cells from ES cells has been listed as top 10 scientific breakout in Science journal, 2004. Actually, the embryonic stem cells/germ cells differentiation platform is not only a good model for reproductive system development but also a novel source for adult germ cells, implicating sufficiently theoretical and practical significance. Particularly as the physiological origin of germ cells, EG cells appear in principle better than ES cells when generation of germ cells is required. In vivo, PGCs can migrate to gonad, amplifying abundantly and initiating meiosis to form germ stem cells. Nevertheless, due to limited samples and absence of specific markers for PGCs, related basic research concerning regulatory mechanisms on PGCs differentiation is rare. In this study, in vitro differentiation of PGCs-derived hEG cells is carefully examined of gene expression profiles, including multipotent stem cell markers Oct4 and Nanog, germ lineage markers Stella and VASA, and meiosis-related molecules SCP1, SCP3, GDF9 and TEKT1.ResultsLIF-transfected human lung fibroblasts as feed layer for human EG cellsLIF, a kind of pleotropic cytokines, is indispensable for maintaining ES cell selfrenewal by suppressing its differentiation. At a first step, the human LIF cDNA was cloned and inserted into the eukaryotic expression vector pcDNA3.0, the pcDNA/lif. Then, embryonic lung fibroblasts (ELFs) with typical fibroblastic morphology as well as high proliferating potential were isolated from lung tissue of human 9-week embryos. Then the ELFs of 5th passages were transfected with pcDNA/lif, and positive clones were collected and expanded after screening with G418 supplement. Stable expression of lif gene was further confirmed by RT-PCR and western blot. Hence, the modified fibroblasts were named hELF/lif.Thereafter, human embryonic gonad cells were transferred to hELF/lif feed layer without addition of recombinant LIF. Upon close observation, PGCs-derived hEG cells were capable of forming canonical nestle-like colonies, which displayed remarkable ALP activity and were stained positive for embryo-specific antibodies (SSEA-1、SSEA-4、TRA-1-60、 TRA-1-81), undifferentiated markers (Oct-4,Nanog and Rex-1) and hTERT. Chromosome analysis of 25-day cultures revealed a normal karyotype. Immunohistochemistry and immunofluorescent examination of 14-day EB showed the expression of mesoderm and endoderm markers, Desmin and AFP respectively.RT-PCR analysis of hEG cells, day 7 EB and day 14 EB demonstrated transcriptional activities of the three germ layers: endoderm (α-FP,Pdx-1);mesoderm (CD34,enolase); and ectoderm (Vimentin,NF68), indicating multilineage potential of hEG cells. Of note, hEG cells cocultured with hELF/lif other than with hELF expressed c-myc, the target gene of LIF signaling cascade.In vitro differentiation of hEG cells to germ cells and related molecular mechanismsUndifferentiated hEG cells displayed significant transcription of Oct4 and Nanog, with the latter immediately and markedly downregulated and eventually abolished upon induced to EB. In contrast, expression of Oct4 was retained at a lower level. Within 7-day EB, expression of STELLA and VASA was detected, in consistence with the in vivo finding. Meanwhile, mRNA of oocyte-specific GDF9 and spermatogonium-specific TEKT1 was observed in 3-day and 7-day EB respectively. Thereafter, meiosis marker SCP1 was also detected. Moreover, Nanog could not be detected in SCP3 positive cells within the EB structures. Subsequently, analysis of 7-week and 13-week embryos was performed. Nanog was abundantly expressed by migrating PGCs. Thus, Nanog might function as an inhibitor of embryonic germ cell differentiation. As decreased expression of Nanog is indispensable for meiosis in vivo, downregulation of Oct4-modulated Nanog appears also critical for hEG cell differentiation in vitro. However, direct evidence is required in the future.ConclusionsHere, we have shown that genetically modified hELF could express exogenous LIF and acted as a novel and appropriate niche for in vitro growth of hEG cells, reflecting promising utilities in further basic research and clinical cell therapies. Furthermore, hEG cells are also capable of being driven into the germ cell pathway as evidenced by a well orchestrated gene expression, highlighting its great potential either as an in vitro model mimicking germ cell ontogeny or as a valuable pool for germ cell preparations to a great deal of patients.更多还原