胚胎干细胞的定向诱导分化及其应用前景

胚胎干细胞(embryonic stem cell,ES细胞)主要来自于胚胎发育早期囊胚中内细胞群(i nner cel l mas s,I CM),具有无限增殖、自我更新和多向分化的特性。理论上可以诱导分化为机体中200多种细胞,可作为细胞移植、组织替代,甚至器官克隆的细胞供体,为将来治疗人类诸多难治性疾病提供细胞来源。本文简述了胚胎干细胞的诱导分化方法、定向分化的一些细胞种类以及其应用前景。     

胚胎干细胞体外诱导分化的研究进展

胚胎干细胞(Embryonic stemcells,ES细胞)是从附植前胚胎的内细胞团(ICM)或附植后胎儿的原始生殖细胞(PGCs)经体外抑制分化培养、分离和克隆得到的具有发育全能性的细胞。ES细胞体外诱导分化的研究自从20世纪90年代开始到现在,一直是生命科学和医药工程领域的研究热点,并且近年来这方面的研究也取得了一些突破性的进展。本文简述了胚胎干细胞的诱导原理、方法和定向分化的一些细胞种类以及目前研究存在的问题和困难。     

胚胎干细胞向生殖细胞分化的研究进展

胚胎干细胞是一类全能型细胞,能在特定条件下诱导产生生殖细胞。近年来,科学家在胚胎干细胞的研究方面取得了一些突破性进展,能够突破常规,治疗生殖细胞缺乏的不孕不育症,对临床治疗和保护濒临灭绝的动物具有重要意义。笔者对胚胎干细胞体外诱导分化为生殖细胞的相关技术进行综述。     

胚胎干细胞向生殖细胞分化的研究进展

生殖细胞来源于原始生殖细胞,其在体内的分化机制已基本清楚。随着生殖细胞研究的不断深入,通过体外诱导途径获得生殖细胞已成为现实。将胚胎干细胞体外分化为上胚层样细胞,进而分化为原始生殖样或原始生殖细胞。将它们迁移入胎儿生殖腺,具有减数分裂及产生精子能力,与卵子结合移入缺生精管的生殖腺的新生鼠可以产生健康后代。为此,体外诱导生殖细胞的成功,进一步阐明了胚胎干细胞向生殖细胞分化的机制,为更有效利用干细胞造福人类奠定了基础。     

胚胎干细胞的体外诱导分化

胚胎干细胞是从早期囊胚内细胞团或桑葚胚中分离的一种多潜能细胞,具有体外无限增殖和自我更新能力,而且还能被定向诱导分化形成机体几乎所有的细胞类型,如肝细胞、造血细胞、成骨细胞、神经细胞、神经胶质细胞、肌肉细胞、胰岛细胞、内皮细胞、黑色素细胞、淋巴细胞、脂肪细胞、成纤维细胞等.     

胚胎干细胞体外诱导分化的研究进展

胚胎干细胞(embryonic stem cell，ES细胞)是由早期胚胎内细胞团(ICM)或原始生殖(PGC)细胞经体外分离、抑制分化培养获得的多潜能细胞。ES细胞具有发育分化的多潜能性和无限的自我更新能力，能在体外长期培养并具有向机体各种组织细胞分化的潜能。由于ES细胞的全能性，在体外可以分化出包括3个胚层在内的所有细胞，     

哺乳动物胚胎干细胞研究进展

胚胎性干细胞包括从动物早期胚胎的卵裂球、囊胚内细胞团细胞分离建系的胚胎干细胞（embryonic stem cell,ESC）、从胚胎生殖嵴原始生殖细胞（primordial germ cell,PGC）分离建系的胚胎生殖细胞（embryonic germ cell,EGC）和来源于畸胎瘤中的胚胎性癌细胞（embryonic carcinoma cell,ECC）。ESC具有发育分化的多潜能性和无限的自我更新能力,能在体外长期培养并具有向机体各种组织细胞分化的潜能。所以,被广泛应用于胚胎发育与细胞分化调控的研究,作为修复器官与器官移植的种子细胞,并可用于转基因动物的生产。作者主要综述了干细胞的最新研究进展,ESC培养扩增诱导机制,定向分化方法。     

胚胎干细胞向神经细胞定向诱导分化方法的研究进展

胚胎干细胞具有自我更新和多向分化潜能,有望成为治疗神经系统疾病重要的种子细胞来源。如何高效地诱导胚胎干细胞向特定神经细胞分化是目前研究的热点。本文就胚胎干细胞定向分化成神经细胞的3种方法:RA诱导法、谱系选择法和SDIA法及其移植研究做一综述。     

小鼠胚胎干细胞向骨骼肌细胞分化的研究进展

近年来,胚胎干细胞的应用越来越广泛,在体外将小鼠胚胎干细胞诱导分化为肌肉细胞,并且利用这些分化得来的肌肉细胞治疗肌肉退行性疾病,一直是胚胎干细胞研究领域的热点,而胚胎干细胞的分化机制更是其中的难点。目前,用于诱导小鼠胚胎干细胞分化为骨骼肌细胞的方法很多,但分化的效率并不是很高,所以研究胚胎干细胞向骨骼肌细胞方向分化的机制显得尤为重要。文章仅就最近几年对小鼠胚胎干细胞向骨骼肌细胞方向分化的一些方法及其机制作一综述。     

不同方法从小鼠胚胎干细胞分化拟胚体

建立简便有效的胚胎干细胞体外分化研究中制备拟胚体的方法。本实验以3种方法形成拟胚体：滋养层过生长法、悬浮法和胶原酶处理法,并与传统的悬滴法进行了比较。在不同的分化培养体系中以最终分化出搏动的心肌细胞评价形成有分化发育能力的拟胚体的效率。以RT—PCR检测了拟胚体和心肌细胞形成中基因的表达。结果表明,4种方法分化出搏动心肌细胞的比例不同。滋养层过生长法形成拟胚体的比例接近悬滴法．而悬浮法和胶原酶处理形成拟胚体的比例显著低于滋养层过生长法和悬滴法。形态学研究显示,拟胚体发育经历了简单拟胚体阶段到囊状拟胚体或成熟拟胚体。在拟胚体成熟过程中特异性胚层分化发育分子标记HNF-4、Bmp-4与Otx-2表达上调,与心肌细胞分化相关的基因α—MHC、β-MHC、GATA-4及Nkx2．5同样表达上调。表明不同途径可获得不同比例的有发育能力的拟胚体,在体外分化研究中滋养层过生长法则是简便有效的获得正常发育拟胚体的方法。     

Melatonin mitigates the adverse effect of hypoxia during myocardial differentiation in mouse embryonic stem cells

BackgroundHypoxia causes oxidative stress and affects cardiovascular function and the programming of cardiovascular disease. Melatonin promotes antioxidant enzymes such as superoxide dismutase, glutathione reductase, glutathione peroxidase, and catalase.ObjectivesThis study aims to investigate the correlation between melatonin and hypoxia induction in cardiomyocytes differentiation.MethodsMouse embryonic stem cells (mESCs) were induced to myocardial differentiation. To demonstrate the influence of melatonin under hypoxia, mESC was pretreated with melatonin and then cultured in hypoxic condition. The cardiac beating ratio of the mESC-derived cardiomyocytes, mRNA and protein expression levels were investigated.ResultsUnder hypoxic condition, the mRNA expression of cardiac-lineage markers (Brachyury, Tbx20, and cTn1) and melatonin receptor (Mtnr1a) was reduced. The mRNA expression of cTn1 and the beating ratio of mESCs increased when melatonin was treated simultaneously with hypoxia, compared to when only exposed to hypoxia. Hypoxia-inducible factor (HIF)-1α protein decreased with melatonin treatment under hypoxia, and Mtnr1a mRNA expression increased. When the cells were exposed to hypoxia with melatonin treatment, the protein expressions of phospho-extracellular signal-related kinase (p-ERK) and Bcl-2-associated X proteins (Bax) decreased, however, the levels of phospho-protein kinase B (p-Akt), phosphatidylinositol 3-kinase (PI3K), B-cell lymphoma 2 (Bcl-2) proteins, and antioxidant enzymes including Cu/Zn-SOD, Mn-SOD, and catalase were increased. Competitive melatonin receptor antagonist luzindole blocked the melatonin-induced effects.ConclusionsThis study demonstrates that hypoxia inhibits cardiomyocytes differentiation and melatonin partially mitigates the adverse effect of hypoxia in myocardial differentiation by regulating apoptosis and oxidative stress through the p-AKT and PI3K pathway.     

Toxic effects of methylmercury,arsanilic acid and danofloxacin on the differentiation of mouse embryonic stem cells into neural cells

This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABA_A-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA_A-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 µM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.     

胚胎干细胞标记基因在大鼠成体干细胞中的表达

为了给组织工程筛选合适的种子细胞,分别对大鼠脂肪间充质干细胞(rat adipose-derived stem cells,rADSCs)、骨髓间充质干细胞(rat bone marrow stromal stem cells,rBMSCs)和骨骼肌卫星细胞(rat skeletal muscle-derived stem cells,rMDSCs)进行胚胎干细胞标记基因Oct-4、Sox-2、Nanog、IL-6和Tert分析,并确定这些基因在这3种细胞中的表达差异性。首先分别应用酶消化法和机械分离法体外分离培养rADSCs、rMDSCs和rBMSCs 3种细胞。其次应用免疫荧光染色检测Oct-4、Sox-2、Nanog、IL-6和Tert在这3种细胞中的表达,然后采用实时定量PCR和Western blot分别在RNA和蛋白质水平定量检测、分析这些基因在不同细胞中的表达量及其差异。免疫荧光染色结果显示这3种细胞中均表达Oct-4、Sox-2、Nanog、IL-6和Tert,但在RNA水平和蛋白质水平均未检测到Nanog的表达,而IL-6和Tert的相对表达量在这3种细胞中均高于Oct-4和Sox-2。研究结果表明,rADSCs、rBMSCs和rMDSCs虽然具有自我更新和多向分化潜能,但是不具有与胚胎干细胞完全相同的特性。     

胚胎干细胞的研究进展及其应用

胚胎干细胞 (ES细胞 )是一类从早期胚胎内细胞团或原始生殖细胞分离和克隆出的、具有发育全能性和多能性的细胞。ES细胞在动物克隆、转基因动物生产、细胞工程、组织工程、临床克隆治疗和发育生物学、遗传学以及制作动物疾病模型等的研究应用中起着重要的作用。文章介绍了胚胎干细胞及其生物学特性 ,国内外研究进展和新动态。阐述了建立干细胞系的技术要点、ES细胞的应用及发展前景     

Effect of bone morphogenetic protein-4 (BMP-4) on adipocyte differentiation from mouse embryonic stem cells

Embryonic stem (ES) cells can differentiate spontaneously into various lineages in vitro. However, spontaneous commitment of ES cells to the adipocyte lineage is rare. In the present study, bone morphogenic protein-4 (BMP-4) is described as a factor inducing adipocyte differentiation from ES cells at a high rate. For this reason, ES-cell-derived embryoid bodies (EBs) in suspension cultures were exposed to different doses of BMP-4 for 5 days before they were plated onto gelatin-coated tissue culture plates. Moreover, the effect of serum-containing and serum-free media in three different combinations was assessed. Plated EBs, stained with Sudan Black and processed for transmission and scanning electron microscopy, were observed daily for adipocyte formation. Treatment with BMP-4 resulted in the appearance of adipocyte clusters in EBs' outgrowth, depending on the doses applied. Early in differentiation, many small fat droplets were observed in adipocytes, while later on they coalesced and formed a few large fat droplets. Adipocyte clusters had a fibrillar and vascular stroma, and each adipocyte was surrounded with a reticular external lamina. Furthermore, the appearance and development of adipocytes and their changes following 2-3 weeks of starvation mimicked live adipose tissue. In fact, understanding the biological activity of growth and differentiation factors is needed to regulate and direct stem cell differentiation to specific cell types in vitro.     

绵羊胚胎肌源性干细胞的生物学特性

为研究绵羊胚胎肌源性干细胞的生物学特性,取30日龄的绵羊胚胎,采用联合酶消化法及差速贴壁法分离培养获得均质的肌源性干细胞,并对其进行一系列生物学特性检测。结果显示,绵羊肌源性干细胞能传代培养至35代以上;免疫荧光染色和RT-PCR鉴定肌源性干细胞表面标记物Sca-1、CD34、CD144和Desmin呈阳性表达;生长曲线和细胞周期结果显示绵羊肌源性干细胞具有较强自我更新能力;通过不同的诱导方法可以将肌源性干细胞成功地诱导为肝样细胞和胰岛样细胞,Schiff染色和双硫腙染色均呈阳性,RT-PCR检测肝样细胞特异性标记物AFP和ALB以及胰岛样细胞特异性标记物Insulin均呈阳性表达,进一步证明了其诱导分化潜能。结果表明,本试验成功地分离培养获得了均质并具有较强自我更新潜能的绵羊肌源性干细胞,这种干细胞具有向肝样细胞和胰岛样细胞分化的潜能,这为临床治疗提供了潜在的理论基础和大量的种子细胞。     

胚胎干细胞定向分化研究进展(上)

胚胎干细胞 (ES细胞 )是由哺乳动物早期胚胎分离克隆的一类未分化二倍体细胞 ,能在体外增殖 ,又能保持未分化状态。在一定条件下 ,ES细胞可向多个方向分化 ,并生成多种功能细胞。ES细胞在遗传上具有可操作性 ,即导入外源基因、标志基因和报告基因 ,诱导基因突变 ,基因打靶或导入额外原有基因使之过度表达等。利用该特性可制作各种实验模型并进行各种基因分析。ES细胞能被诱导分化为构成机体的任一种细胞类型 ,可用于临床细胞、组织和器官的修复和移植治疗 ,研究ES细胞的定向分化有着重要的理论和应用价值。1　ES细胞分化机制研究现状目…     

胚胎干细胞定向分化研究进展(下)

(续上期 )尽管在造血生长因子的刺激及基质细胞存在的情况下 ,ES细胞可以向造血细胞分化 ,但是离临床应用还有相当长的距离。如何诱导ES细胞高效地向某一系造血细胞或血液细胞分化的方法还不成熟。比如ES细胞在体外能分化形成淋巴细胞 ,但效率很低 ,只有少数拟胚体 (EB)中含有淋巴细胞 ,表明诱导ES细胞分化为淋巴细胞尚需补充一些其他的未知因子。而且要尽可能模拟体内造血生成的环境。2 2　向神经细胞的定向分化　在体外培养中 ,用全反式维甲酸 (RA)能诱导ES细胞向神经细胞分化 ,可形成有典型神经冲动等电生理反应的神经细胞 ,神经…