人胚胎干细胞饲养层培养体系的研究进展

人胚胎干细胞(hES细胞)来源于着床前人囊胚内细胞团(ICM),由于具有体外无限增殖和分化成3个胚层来源的各种细胞的潜能,使其成为当今生命科学的研究热点.建立一个理想的hES细胞培养体系是利用它的前提.目前,最常用的hES细胞的体外培养方式是将其培养在饲养层细胞上.迄今为止,已经有多种细胞用于hES细胞的体外培养.饲养...     

人胚胎干细胞无血清无饲养层培养体系的建立

目的 拟建立人胚胎干细胞无血清无饲养层的培养体系.方法 将干细胞分别置于Knockout培养基(第1组)、自制无血清培养基(第2组)和添加bFGF及肝素的改良无血清培养基(第3组)中培养25代后观察比较3组干细胞形态学变化、克隆数目及大小.并借助细胞免疫组化、流式细胞术和体外拟胚体形成等方法检测干细胞的未分化状态和全能性.结果 传代25次后,第2组克隆逐渐分化.第1组和第3组的干细胞均呈典型的人胚胎干细胞形态特征;细胞表达Nanog,Oct-4,Tra-1-60,SSEA-4,但不表达SSEA-1;流式检测也显示SSEA-4高表达,SSEA-1低表达;体外分化能形成拟胚体.通过单位视野下克隆个数及大小的比较,结果显示第2组与第1组和第3组间的差异显著.结论 本研究通过改良本实验室已获得国家专利的无血清培养基,初步建立了人胚胎干细胞无血清无饲养层培养体系,培养效果与公认培养体系无明显差异.     

5种不同人源性饲养层对人胚胎干细胞生长支持情况的研究

目的比较5种不同人源性饲养层对人胚胎干细胞生长支持的情况。方法经签署相关知情同意书后,按人源性饲养层的标准化操作规范获得包皮、减胎、胎儿皮肤、输卵管、睾丸组织的成纤维细胞,经抗波形白荧光染色鉴定,扩增培养后传代并冻存,经丝C裂霉素处理,将胚胎干细胞NS-1接种于其上,观察NS-1贴壁生长情况,AKP染色计算NS-1分化率。结果获得了5种人源性饲养层,分别为包皮、减胎、输卵管、胎儿皮肤,睾丸组织的成纤维细胞,经抗波形蛋白鉴定,阳性表达,抗角型蛋白呈阴性。5种人源性饲养层在hESCs的贴壁率及生长率上与MEF均无统计学差异,但在长期的培养过程中,MEF支持的hESCs未分化率明显高于5种人源性饲养层支持的hESCs未分化率。结论在常规的培养系统中,5种人源性饲养层不能长期支持人胚胎干细胞的生长。     

小鼠胚胎干细胞饲养层培养体系的优化筛选

目的：建立小鼠胚胎干细胞饲养层培养体系。方法：用5种不同鼠胚成纤维细胞为饲养层，进行小鼠胚胎干细胞的分离培养，观察5种饲养层培养体系对小鼠胚泡发育，内细胞团增殖及胚胎干细胞分离培养的作用。结果：原代或冻存复苏后的原代培养鼠胚成纤维细胞用于制备饲养层，有利于胚泡的贴壁，孵化，内细胞团增殖形成巢式生长集落，离散后培养，可以观察到胚胎干细胞集落的出现，并可在短期内维持胚胎干细胞的正常形态，不发生分化，与其他三组有明显差异。结论：原代或冻存复苏后的原代培养鼠胚成纤维细胞饲养层是用于胚胎干细胞分离培养的有效的培养体系。     

人羊膜间充质干细胞无血清培养方法的建立

目的 建立一种人羊膜间充质干细胞(hAMSCs)的无血清培养方法.方法 用胰蛋白酶-胶原蛋白酶两步消化法分离人羊膜间充质干细胞,用含1×ITS、0.5％人血清白蛋白、10 ng/mL bFGF、100μg/mL L-ascorbic acid、100 U/mL青链霉素及2.5μg/mL两性霉素B的DMEM/F12无血清...     

人胚胎干细胞的培养条件与鉴别的方法学研究

目的：研究人胚肺成纤维细胞(human Embryonic Lung Fibroblast，HELF)饲养层对人胚胎干细胞(Human Embryonic Stem Cells，hES细胞)生长的作用，摸索适宜的hES细胞培养与识别方法。方法：以流产胎儿肺组织制备HELF饲养层，取生殖嵴或背肠系膜及其周围组织，获得由人原始生殖细胞(human Primordial Germ Cells，hPGCs)转化成的hES细胞。比较其在不同条件下的增殖行为，并通过生物学特性鉴别该细胞系。结果：HELF饲养层在LIF存在的条件下有利于支持hES细胞的体外生长；hES细胞碱性磷酸酶组织化学染色及端粒酶ELISA检测阳性；具有正常的核型；在体外发展为类胚体。结论：HELF饲养层和LIF的协同作用能支持hES细胞的生长；建立通过生物学特性鉴别hES细胞的方法。     

人胚胎干细胞在血清和无血清培养体系中的特性比较

目的 探讨血清培养体系和无血清培养体系对人胚胎干细胞（hES cells）生长特性的影响。 方法 将人胚胎干细胞株BG02接种在丝裂霉素C处理灭活的小鼠胚胎成纤维细胞饲养层上,分别在含血清hES完全培养基或无血清培养基中连续培养25~30代。在不同培养体系下比较人胚胎干细胞形态、集落贴壁率;运用BrdU掺入法检测人胚胎干细胞增殖,用细胞计数法计算细胞倍增时间;采用免疫荧光染色检测人胚胎干细胞特异性分子标志的表达;用流式细胞仪检测人胚胎干细胞Oct-4,Nanog阳性的比例;RT-PCR检测成纤维细胞生长因子(FGFs)家族基因的表达。 结果 在两种培养体系的BG02细胞都具有人胚胎干细胞的形态特征。在含血清培养体系下,BG02细胞集落贴壁率和表达OCT-4,Nanog的阳性细胞明显高于无血清培养体系( P<0.05)。无血清培养体系中,BG02细胞生长速度明显高于含血清培养体系,细胞倍增时间分别为(33.8±4.3) h、(45.9±5.7) h,( P<0.05)。无血清培养体系的BG02细胞高表达 FGF2 、 FGFR2 、 FGFR4 。 结论 两种不同培养体系中人胚胎干细胞的体外培养特性存在一定的差异,可能与BG02细胞 FGFs 家族基因激活有关。     

大鼠骨髓胚胎样干细胞的培养和鉴定

目的:探索骨髓胚胎样干细胞(ELSCs)的分离纯化和鉴定方法,并探讨该细胞分离培养方法的可行性。方法:采用全骨髓培养、明胶包被和无血清培养的方法,培养SD大鼠骨髓细胞,5 d后进行第1次传代,以后每2 d传代1次。观察其形态结构;免疫荧光检测其表面标志物CD73、CD90、CD105、SSEA-4、Sox-2、Nanog;流式细胞术检测干性标志物SSEA-4、OCT-4、Sox-2、Nanog的表达,并与骨髓间充质干细胞(MSCs)相比较;检测其向成骨细胞和成脂细胞的诱导分化能力。结果:获得的ELSC呈现长梭形,体积小且形态均一。免疫荧光示其表达CD73、CD90、CD105、SSEA-4、Sox-2、Nanog;流式细胞术检测示ELSCs表达CD73、CD90、CD105,且纯度高于95%,与MSCs相比,ELSCs的SSEA-4、OCT-4、Sox-2、Nanog等干性基因表达较高。结论:该方法获得的ELSCs干性较强、强度较高,可分离、培养出ELSCs。     

人胚胎干细胞体外培养和扩增的研究进展

体外维持人胚胎干细胞未分化的增殖状态受细胞生长环境和内源性调节因子的控制。本文阐述了目前体外培养和扩增人胚胎干细胞细胞的各种系统,分析了影响细胞增殖和不分化的因素及其信号转导机制,指出了为临床细胞替代治疗疾病提供细胞来源的人胚胎干细胞培养系统的发展趋势。     

维持人胚胎干细胞多能性顺式作用元件的筛选

目的 在人胚胎干细胞(hESCs)中建立一种可行的CRISPR文库筛选方法,筛选参与维持hESCs多能性的顺式作用元件.方法 将诱导型Cas9蛋白表达元件插入hESC基因组中,构建诱导型Cas9稳定表达株(iCas9-hESC).通过分析已发表的hESCs中的染色质互作信息,确定可能参与多能性基因调控的顺式作用元件,并...     

人胚胎干细胞向内皮细胞的分化诱导

BACKGROUND:Human embryonic stem cells exhibit self-renewal and multi-differentiation potential, and can differentiate into endothelial cells under certain induction conditions. OBJECTIVE:To explore induced conditions of the human embryonic stem cells differentiating into endothelial cells and to investigate the effect of vascular endothelial growth factors on the endothelial differentiation of human embryonic stem cells. METHODS:After resuscitation, passage 40 human embryonic stem cell lines H9 were subjected to suspension culture to prepare embryos, and after 5-day culture, these cells were cultured in attachment medium to differentiate into embryoid bodies, followed by induction with 50 µg/L vascular endothelial growth factors. Passage 2 and 15 embryonic stem cells after induced differentiation were taken for Dil-Ac-LDL uptake test and immunohistochemical staining, respectively. RESULTS AND CONCLUSION:After 1-day culture, cord-like or polygonal monolayer cells around embryoid bodies showed bud-like and radial growth with a relative rapid speed merging into surrounding colonies; at 2-3 days, the number of suspension cells increased further, but the small-round cells in the center began to die; at 5 days, embryoid bodies started to passage, and aggregated cells exhibited typical paving stone-like appearance. Moreover, some human embryonic cells after induced differentiation could actively take up fluorescent labeled LDL, and red fluorescent particles appeared. Additionally, passage 15 embryonic stem cells after induced differentiation could express CD31 and FLK-1. These findings suggest that human embryonic stem cells induced by vascular endothelial growth factors can differentiate into endothelial cells.     

Nanog慢病毒载体构建及在胚胎干细胞分化调控中的应用

BACKGROUND:There is a lack of safe and effective modular lentivectors in differentiation regulation of embryonic stem cells. OBJECTIVE:To construct a modular lentivector, Puro-Nanog-hrGFP, with Nanog promoter-controlled humanized renilla reniformis green fluorescent protein (hrGFP) and puromycin and to observe the expression of Nanog during the differentiation of Nanog-hrGFP-transfected mouse embryonic stem cell lines. METHODS:After PCR amplification, Nanog, hrGFP and entry vector were recombined into the pDest-puro vectors to generate the lentiviral expression vector, Puro-Nanog-hrGFP, by the LR reaction. Through lentivirus production, transduction and puromycin screening, the transduced cell lines with Nanog-hrGFP gene were generated and identified. Expression of Nanog during the differentiation of transgenic mouse cell lines was detected. RESULTS AND CONCLUSION:The lentiviral expression vectors Puro-Nanog-hrGFP were constructed successfully by Gateway technology, and then the transducted cell lines were obtained by lentiviral infection. The expression of Nanog was gradually decreased during the process of transgenic cell lines differentiation, which provides a new tool for further investigation on regulation of stem cell differentiation.     

小鼠胚胎干细胞不同分化阶段UGT1a1、UGT1a6和mGST1的表达特征

BACKGROUND:Until now, limited information has been neported on the characterization of udines 5-diphosphateglucuronosyl transferase (UGT) 1a1, UGT1a6, and microsomal glutathione S-transferase 1 (mGST1) during the differentiation of mouse embryonic stem cells. OBJECTIVE:To observe the expression characteristics of UGT1a1, UGT1a6 and mGST1 at different stages of differentiation of mouse embryonic stem cells. METHODS:Embryonic fibroblasts from Wistar rats were isolated and cultured as feeder cells. Mouse embryonic stem cells seeded onto these feeder cells were induced to differentiate into hepatocytes. Subsequently, expression of UGT1a1, UGT1a6 and mGST1 was detected using western blot assay, and catalytic activity of mGST1 was determined by high performance liquid chromatography. RESULTS AND CONCLUSION:Increasing UGT1a1 expression was visible during the whole cell differentiation, while UGT1a6 exhibited no expression initially but a higher level at 18 days of differentiation. mGST1 expression was visible at high level throughout the differentiation, but its expressive abundance was still lower than that in the adult mouse stem cells. At 18 days after the beginning of differentiation, the catalytic activity of mGST1 in microsomal liver tissue was 7.65 μmol/(min•g). Taken together, stabilized UGT1a1, increased UGT1a6 and little mGST1 expression are confirmed in the differentiation of embryonic stem cells into hepatocytes.     

不同培养方法对人脐带间充质干细胞的影响

BACKGROUND:Umbilical cord stem cells mainly derive from full-term infants, and common culture methods include tissue-attached method and trypsin-digestion mehod. However, effects of different culture methods on the separation of umbilical cord mesenchymal stem cells remain many disputes. OBJECTIVE:To observe the effects of different culture methods on umbilical cord mesenchymal stem cells. METHODS:Umbilical cords of 30 healthy full-term and caesarean delivery infants were selected, and cultured using tissue-attached method or trypsin-digestion method to isloate and culture human umbilical cord mesenchymal stem cells. Meanwhile, cell growth was measured by flow cytometry. RESULTS AND CONCLUSION:The fusiform-shaped cells began to separate from the umbilical cord tissue that was primary cultured using tissue-attached method, and 10 days later, the cell fusion reached 80%; after the umbilical cord was cultured using collagenase-trypsin digestion for 5 days, a small amount of adherent cells with different shapes appeared, and the fiber-like cells reached 80% of confluence until 2-week culture. There was no significant difference in the growth of umbilical cord mesenchymal stem cells cultured by different culture methods (P > 0.05). Moreover, cells cultured by two methods were all positive for CD13, CD29, CD44 and CD105. These results demonstrate that human umbilical cord mesenchymal stem cells exhibit a high success rate in primary culture using tissue-attached method, which is superior to the trypsin-digestion method.     

缺氧诱导骨髓间充质干细胞条件培养基体外修复辐射损伤小肠上皮细胞变化

背景：间充质干细胞条件培养基含有丰富的间充质干细胞旁分泌物质,被认为是干细胞移植理想的替代方案。然而,正常状态的间充质干细胞条件培养基可能由于旁分泌活性不足,常无法有效修复损伤组织。 目的：观察缺氧诱导的骨髓间充质干细胞条件培养基(MSC-CMHyp)对大鼠小肠隐窝上皮IEC-6细胞辐射损伤后增殖和凋亡的影响,并进一步探讨其修复小肠上皮细胞的旁分泌机制。 方法：IEC-6细胞接受10 Gy X射线照射后分别加入MSC-CMHyp、正常培养的骨髓间充质干细胞条件培养基(MSC-CMNor组)及DMEM-F12培养基(DMEM-F12组)培养。 结果与结论：锥虫蓝染色、流式细胞仪、Western blot检测结果显示,与DMEM-F12组相比,MSC-CMHyp组IEC-6细胞存活数和增殖率明显增加(P < 0.05),凋亡率和凋亡相关因子Caspases-3/8表达明显下降(P < 0.05),而MSC-CMNor组的各项指标与DMEM-F12组比较差异无显著性意义(P > 0.05)。EILSA检测结果显示,与MSC-CMNor相比,MSC-CMHyp中血管内皮生长因子、碱性成纤维细胞生长因子、胰岛素样生长因子1、和白细胞介素10的含量明显升高(P < 0.05)。结果表明,缺氧诱导的骨髓间充质干细胞条件培养基中细胞因子含量明显升高,显著促进辐射损伤IEC-6细胞的增殖,同时下调细胞凋亡信号,从而进一步促进损伤细胞的修复。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

胚胎体外培养及胚胎干细胞系的建立

背景：人类胚胎干细胞体外建系成功,对人类胚胎发育机制和发育生物学研究、细胞和组织移植治疗某些疾病等领域都有重大意义。目的：综述近年来关于胚胎体外培养及建立胚胎干细胞系的研究进展,重点探讨胚胎体外培养影响因素、人废弃胚胎培养分离内细胞团建立胚胎干细胞系的方法及建立胚胎干细胞系的条件。方法：以“胚胎(embryo),胚胎干细胞(embryonic stem cell),共培养(co culture),序贯培养(sequential culture)”为检索词,由第一作者检索2000至2014年CNKI数据库和SCI数据库,获取有关胚胎体外培养、移植及胚胎干细胞建系的相关文献,并进行系统评价,最终保留58篇文献进行分析。结果与结论：胚胎体外培养条件是影响胚胎移植结局的重要因素,其中包括培养液的成分和培养体系。在过去的研究过程中,培养液的构成及应用已经发生了很大的变化,培养体系也从单一培养发展到共培养、序贯培养。伦理问题及胚胎来源的限制束缚着人胚胎干细胞系的建立,利用临床废弃的低质量的胚胎可作为建立人胚胎干细胞系的材料来源之一,有效的缓解了建立人胚胎干细胞系过程中胚胎缺乏的问题,并减少其中的伦理学纷争。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人原代胃癌细胞中肿瘤干细胞的分化培养

BACKGROUND:There is a close relationship between tumor stem cells and tumor occurrence and recurrence, but there are still some disputes in the presence of tumor stem cells in all tumors. OBJECTIVE:To investigate the differentiation and culture of tumor stem cells in human primary gastric cancer cells. METHODS:Primary gastric cancer cells isolated from fresh gastric cancer tissues were stained with hematoxylin-eosin and identified by immunohistochemical detection of carcinoembryonic antigen. The CD44 expression of the cells was detected using immunofluorescence method. Magnetic activated cell sorting was used to isolate CD44⁺ gastric cancer cells that were then seeded subcutaneously behind the armpit of mice. Growth of implanted tumor cells was observed. RESULTS AND CONCLUSION:Human primary gastric cancer cells were isolated in serum-free medium. Compared with the routine culture group, the number of CD44⁺ cells (P < 0.05) and the tumor volume were significantly increased in the spheroid culture group. Furthermore, at 90 days after transplantation, the tumor volume of mice in spheroid culture group was significantly higher than that in the routine culture group. These experimental findings indicate that gastric cancer cells with certain tumorigenicity can be successfully isolated from gastric cancer cells using serum-free culture method and magnetic activated cell sorting method.     

人胚胎干细胞建系培养及体外诱导分化的研究进展

人胚胎干细胞具有发育全能性,在特定条件下能分化成多种类型的细胞.人胚胎干细胞的研究对人胚胎发育机制、人基因功能研究和治疗性克隆有着重大的意义.本文从人胚胎干细胞建系、培养及体外诱导分化等方面作一综述.     

三维培养诱导人脂肪间充质干细胞微球的成软骨分化能力

背景：关节软骨损伤后修复结果不满意,需要新的手段,而脂肪间充质干细胞较适宜做种子细胞诱导软骨,然而怎么能够使诱导的软骨具有功能需要研究。 目的：采用三维培养体系诱导人脂肪间充质干细胞微球向软骨分化。 方法：无菌切取吸脂术后脂肪组织,分离培养人脂肪间充质干细胞,传至第3代进行流式细胞术分析,成骨成脂肪诱导等鉴定,同时也给予合适的培养条件用三维培养的方式向软骨细胞诱导,并行阿利辛蓝染色鉴定糖胺多糖的合成,苏木精-伊红染色进行组织学分析,免疫荧光检测Ⅱ型胶原表达,称质量测量软骨硬度。 结果与结论：分离的人脂肪间充质干细胞CD105,CD44,CD29均高表达,而 CD45,CD34低表达,并且成骨成脂诱导后细胞茜素红染色和油红O染色均为阳性。三维培养法诱导的软骨细胞可表达大量糖胺多糖及Ⅱ型胶原。结果证实,三维培养法诱导人脂肪间充质细胞向软骨分化后,具有软骨细胞的特性。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人髌下脂肪垫来源脂肪间充质干细胞的分离、培养及鉴定

背景：髌下脂肪垫在膝关节手术中经常要部分切除,其可以作为脂肪间充质干细胞的重要来源。 目的：探讨自髌下脂肪垫中分离、培养脂肪间充质干细胞的策略及细胞分子表面标记情况。 方法：髌下脂肪垫组织取自膝关节镜手术的患者,以Ⅰ型胶原酶消化消化脂肪组织获取干细胞,用10%低糖DMEM培养基培养,利用MTT法测定不同代细胞增殖情况并绘制生长曲线。检测第5代细胞表面CD29及CD44的表达。 结果与结论：培养24 h后可见原代细胞贴壁,1周后细胞呈纺锤型并且增殖速度加快,传代后的细胞贴壁及增殖细胞速度加快。生长曲线示第2及第5代的细胞增殖能力明显较第8代能力强。所取细胞能够分化为骨细胞和脂肪细胞。流式细胞仪检测结果显示第5代脂肪间充质干细胞重96.8%表达CD29,97.6%表达CD44。提示自髌下脂肪垫分离及提取脂肪干细胞简单易行,所得细胞的纯度及增殖能力均符合组织工程种子细胞的基本条件。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程