摘要
背景及目的：胚胎干细胞(embryonic stem cell, ESC)是指从早期胚胎内细胞团或原始生殖细胞分离出来的一种具有无限增殖能力和全方向分化能力的一种多潜能细胞,具有修复甚至替换丧失功能的组织和器官的潜在应用价值。如何诱导胚胎干细胞向特定细胞类型的分化是其基础研究和临床治疗应用的关键点。胚胎干细胞的这种定向分化能力除了细胞内各种转录因子的内在决定因素外,细胞间因子的分化诱导、抑制作用及细胞外物质的介导作用等外源性因素也是必不可少的。目前,在胚胎干细胞的众多诱导分化方式中,采用共培养的方式来诱导其定向分化已经成为组织工程领域一种新兴而很有发展前景的技术,在很多医学领域都有成功的报道,如心血管、呼吸系统和肝脏等领域。在泌尿系统领域,既往研究发现胚胎干细胞与离体的胎肾器官共培养能成功诱导其向肾系细胞分化。但是有关胚胎干细胞在体外能否通过和一种特定的细胞相互作用继而定向分化成肾系细胞并未见报道。为此本实验尝试体外通过Transwell双层培养皿将胚胎干细胞同大鼠胚胎后肾间充质细胞(metanephric mesenchymal cells MMCs)进行共培养的方法,观察共培养条件下,胚胎干细胞能否定向分化成肾脏样细胞,期望为下一步应用于急慢性肾功能衰竭的动物实验模型的治疗奠定基础。
方法：1.取妊娠13.5d的胎鼠,采用组织消化法分离培养出小鼠胚胎成纤维细胞(Mouse Embryonic Fibroblasts MEFs),对MEFs的生长形态、生长曲线及分裂指数进行观察,MTT法筛选丝裂霉素C (Mytomycin C, MMC)作用的最佳浓度和时间。
2.收集昆明系小鼠3.5d的囊胚,培养在经MMC处理的第三代小鼠胚胎成纤维细胞饲养层上,对分离克隆出的胚胎干细胞集落的形态学、碱性磷酸酶(Alkaline phosphatase, AKP)、全能型因子(OCT-4,NANOG)的表达、核型分析以及体内外分化能力加以鉴定。
3.从孕12.5-14.5d的SD胎鼠中分离培养出后肾间充质细胞,并对其进行形态学、细胞免疫学和电镜的鉴定。
4.采用直接悬浮培养法使小鼠胚胎干细胞形成拟胚体(EBs)后,将拟胚体细胞与胚胎后肾间充质细胞通过Transwell培养系统共培养7d,RT-PCR检测共培养的拟胚体细胞是否表达肾脏发育相关的特征基因(WT-1,Wnt-4, pax-2,c-Ret)和终末分化的肾细胞标志物(podocalyxin, Nephrin)。
结果：1.MEFs为一种贴壁生长且增殖速度较快的细胞,第三代细胞增殖旺盛,第5代以后细胞开始变形并趋于衰老。MMC能抑制胚胎成纤维细胞的增殖,最佳的作用浓度和时间是10ug／ml作用2.5-4.0h,20ug/ml作用1.0-2.5h。
2.分离得到的ES细胞有典型的形态学特征：集落呈鸟巢状,边缘清楚结构致密,隆起生长；碱性磷酸酶染色呈强阳性；RT-PCR分析显示OCT-4,NANOG等全能性因子表达阳性；具有正常的二倍体核型；可形成拟胚体；皮下注射到免疫缺陷小鼠可形成包括三个胚层的畸胎瘤。
3.SD大鼠胚胎后肾间充质细胞为一种贴壁生长、生长速度较快、呈漩涡样生长的细胞。细胞体积较小,胞浆少、胞核大、核仁明显。间质细胞标志物波形蛋白阳性,而上皮细胞标志物角蛋白为阴性。电镜下细胞呈不规形,胞核大,内有细胞器,细胞表面有微绒毛。
4.拟胚体细胞与胚胎后肾间充质细胞共培养7d后,通过RT-PCR检测到共培养后的胚胎干细胞表达肾脏发育相关标志物(WT-1, Wnt-4,pax-2, c-Ret)和终末分化的肾细胞标志物(podocalyxin, Nephrin)。
结论：采用共培养模型,大鼠胚胎后肾间充质细胞可以诱导昆明小鼠胚胎干细胞定向分化为肾脏样细胞。但是,分化得到的这种细胞仅仅是一种前祖细胞,而非一种特定的肾脏细胞。因此如何得到一种特定的肾脏细胞,以及任何将分化后的细胞从未分化的胚胎干细胞分离出来进行纯化从而应用于急慢性肾功能衰竭动物实验模型的治疗中将是我们下一步要研究的关键问题。
Background and Objective:Embryonic stem cell is a pluripotent cell derived from the inner cell mass of early mammalian embryo or primordial germ cells. It is capable of unlimited, undifferentiated and proliferative in vitro. It has potential applications in treating a wide array of diseases in the brain, kidney, bone and many other tissues. So the key point of embryonic stem cell in basic research and clinical application is how to induce it into a specific cell line. The orientation of embryonic stem cell is in addition to cellular differentiation of various definitive transcription factors, extracellular material inhibition mediated by endogenous factors is also essential. Now, inducing embryonic stem cell differentiation in vitro by co-culture hasbeen a very emerging and promising technology in tissue engineering. It has success reports in many medical fields, such as in the field of cardiovascular, respiratory and liver. In the field of urinary system, the recent researches showed that embry onic stem cell could differentiate into a renal lineage via embryoid bodies. But could embryonic stem cell differentiate into renal-like cell types by co-culturing with a particular type of cell had not reported yet.So we developed an efficient method by which embryonic stem cell could be induced to differentiate into a renal lineage especially the renal ancester cells by co-culturing with the rat meta-nephric mesenchymal cells in Transwell system, which will lay the foundation for the next step in the progression of future renal therapy by the induced renal-like cells.
Methods:(1) The mouse embryonic fibroblast cells (MEFs) were isolated from the mouse embryos of 13.5 day gestation through primary tissue digestion. The morpholo-gy, growth curve and Mitotic Index of MEFs in vitro was investigated. The optional concentration of mitomycin C and the time treated with mitomycin C on MEFs were determined by MTT assay.
(2) The blastocysts (4 dpc) from Kunming species mouse were collected, cultured on the third passages of embryonic fibroblast cell feeder layers treated with mitomycin C. The mESC cells which were pluripotential cells had been identified by morphology of cells, alkaline phosphatase staining, expression of OCT-4 and NANOG by RT-PCR, karyotype analysis, the formation of embryoid body and teratoma which are typical characteristics of ES cells.
(3) The rat metanephric mesenchymal cells were isolated and cultured from the Sprague Dawley rats embryos of 13.5 day gestation. The cells had been identifi-ed by morphology of cells, immunocytology and electron-microscope.
(4) ESCs were induced to form embryoid bodies by suspension culture. The cells from embryoid bodies were co-cultured with metanephric mesenchymal cells in Transwell system for differentiation into renal lineage. The treated ESCs were detected by expressing marker molecules characteristic for initiation of nephrogenesis (WT-1,Wnt-4,pax-2,c-Ret) and terminally differentiated renal cell types (podocalyxin, Nephrin).
Result:(1)MEFs in vitro was a kind of adherent cell with good ability for proliferation,especially in passage 3, but from the 5th passage, MEFs was going to be senescence and distortion. The proliferation of MEFs could be efficiently repressed by mitomycin C.When mitomycin C concentrated on 10ug/ml and acted for 2.5-4 hours or on 20ug/ml and acted for 1-2.5hours, it worked best to inhibit the proliferation of MEFs.
(2)Most of these cells had typical characteristics of mESC.These cells aggregated to form colonies with a clear borderline between colonies and feeder layer cells. The Colonies showed the high activity of AKP, expressed OCT-4 and NANOG by RT-PCR and had a normal karyotype. They also formed embryoid bodies by non-adherent culture in vitro and formed teratoma in severe combined immunodeficiency (SCID) mice in vivo.
(3) The rat metanephric mesenchymal cells are cells with growing adherencely.They growed fast and showed spiral-like growth. The cells were small and they had little cytoplasm, big nucleus and prominent nucleoli.They were vimentin positive and keratin negative. In electron-mieroscope, the cells were irregular with big nuclei, cellular organelle inside and microvilli.
(4) By RT-PCR analysis, after 7 days of co-culturing with metanephric mesenchymal cells, the embryoid body cells gave rise to the cell population expressing the above marker molecules characteristic for nephrogenesis. Conclusion:Embryonic stem cell could be induced into renal-like cells by co-culturing with metanephric mesenchymal cells. However, the induced cells were merely pre-progenitor cells, rather than specific renal cells. Therefore, how to get a specific kind of kidney cells and purify these cells in order to apply to in the treatment of renal repair in experimental models of acute and chronic renal failure is our next step.
引文
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