小鼠胚胎干细胞诱导分化为肾脏样细胞的实验研究

发布时间：2018-02-16 06:24

  本文关键词： 胚胎干细胞 胚胎后肾间充质细胞 共培养 肾发生 分化　出处：《广西医科大学》2010年硕士论文　论文类型：学位论文

【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。
[Abstract]:Background and purpose: embryonic stem cells (embryonic stem cell, ESC) is separated from the inner cell mass of early embryos or primordial germ cells which proliferate and differentiate the ability of a pluripotent cells, tissues and organs with repair or replace the loss of function of the potential value. How to induce embryonic stem cells to differentiate into specific cell types is the key point of the basic research and clinical application. The orientation of embryonic stem cell differentiation in addition to internal factors of various transcription factors in cell differentiation, cell factor induced inhibition of extracellular substances mediated by exogenous factors is also essential at present, in embryonic stem cell differentiation in many ways, the co culture way to induce the differentiation of tissue engineering has become a The emerging and promising technology, has been reported in many medical fields such as cardiovascular, respiratory and liver and other fields. In the field of urinary system, previous studies have found that cells and isolated fetal kidney were cultured successfully induce the differentiation of embryonic stem cells into the kidney. But the embryonic stem cells in vitro and whether through a specific interaction between cells differentiate into renal cells and have not been reported. Therefore this experiment in vitro by Transwell double culture embryonic stem cells with rat metanephric mesenchymal cells (metanephric mesenchymal cells MMCs) by co culture method, co culture observation conditions that embryonic stem cells can differentiate into renal like cells, expected to lay the foundation treatment for experimental animal model for the next step in the application of acute and chronic renal failure.
Methods: 1. pregnant 13.5d fetal rats by tissue digestion of isolated mouse embryonic fibroblasts (Mouse Embryonic, Fibroblasts MEFs) on MEFs morphology, growth curve and mitotic index were observed by MTT screening of mitomycin C (Mytomycin C MMC) the optimal concentration and time.
2. Kunming mice were collected 3.5D blastocysts cultured fibroblast feeder cells in MMC treated mice on the third generation of cloned embryos, embryonic stem cell colony morphology, alkaline phosphatase (Alkaline phosphatase, AKP), universal factors (OCT-4, NANOG) expression, karyotype analysis and in vitro and in vivo the differentiation ability were identified.
3. the mesrenal mesenchyme cells were isolated and cultured from the pregnant 12.5-14.5d SD fetal rats, and their morphology, cell immunology and electron microscopy were identified.
4. by direct suspension culture method of mouse embryonic stem cells formed embryoid bodies (EBs), the embryoid body cells and rimm-18 cells cultured by Transwell co culture systems of 7D, RT-PCR detection of co cultured to whether embryo cells expression of kidney development characteristic base related (WT-1, Wnt-4, Pax-2, c-Ret) and terminally differentiated renal cell markers (podocalyxin, Nephrin).
缁撴灉锛，

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