昆明小鼠胚胎干细胞滋养层制备条件的实验研究

目的:建立小鼠胚胎成纤维细胞(MEFS)滋养层,用于昆明小鼠胚胎干细胞的培养。方法:取妊娠13.5的胎鼠,采用组织消化法分离培养出原代成纤维细胞,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(MMC)作用的最佳浓度和时间;取妊娠3.5d的囊胚在经MMC处理的饲养层上培养,观察胚胎干细胞集落生成情况。结果: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2.5～4h,20ug/ml作用1-2.5h。妊娠3.5d小鼠囊胚在饲养层上培养能形成典型的"鸟巢"状干细胞集落,并可维持胚胎干细胞的正常形态且不发生分化。结论:这种方法制备的滋养细胞层适用于胚胎干细胞的培养。     

提高人胚胎干细胞建系率的优选培养体系与方法

目的:建立一种从废弃胚胎中提高囊胚形成率和质量的培养体系,寻找多种促进内细胞团(ICM)数目增多、贴壁、增值的方法,提高人胚胎干细胞(human embryonic stem cell,hESC)建系效率,建立人胚胎干细胞库。方法:将179枚IVFDay3废弃的胚胎放入优选培养体系中培养(G2.5培养液中添加10%人血清蛋白,人白细胞抑制生长因子(hLIF),碱性成纤维细胞生长因子(bFGF))。到Day7将形成的囊胚全部用机械法分离ICM,接种于丝裂霉素C灭活处理的原代小鼠胚胎成纤维细胞(MEF)上,培养8-9天,每4-5天传代1次。结果:优选培养体系的囊胚形成率为29.1%(52/179),其中A级囊胚形成率为11.2%(20/179),50个ICM贴壁生长,20个出现克隆形态,成功建立11株hESC(FY-hES-11至FY-hES-21)。11株hESC均具有共同的多能性生物学特性。结论:优选培养体系可以明显提高囊胚形成的质量,促进ICM的增值,纯熟的机械切割法可以避免损伤ICM并提高其贴壁率,原代灭活的MEF饲养层可以明显促进细胞增殖。     

昆明白小鼠胚胎干细胞分离与体外培养

为探索昆明白小鼠胚胎干细胞建系方法,将受孕4.5天的昆明白小鼠囊胚用免疫手术法去除滋胚层,然后将内细胞团(ICM)接种于胎鼠成纤维细胞饲养层上培养,形成的胚胎干细胞样集落用胰蛋白酶-EDTA消化法传代,培养后进行相差显微镜观察及碱性磷酸酶染色。结果饲养层上生长的ICM细胞呈典型的ES样细胞集落,传至第8代碱性磷酸酶染色呈强阳性。实验表明免疫手术法适用于昆明白小鼠ES细胞建系,获得的细胞集落具有ES细胞的主要生物学性状。     

建立小鼠胚胎多能干细胞系（ES细胞系）的几个主要影响因素

本文系统地比较了影响建立小鼠胚胎多能干细胞系的几个主要因素。对825个胚的实验进行了分析,其结果表明:延迟囊胚的主要作用是有助于内细胞团(ICM)的增殖;高糖的DMEM有助于囊胚贴壁及ICM的增殖;不含丙酮酸钠的DMEM对ES细胞的集落形成和生长是有利的;饲养层对ES细胞的建系和培养是十分重要的,以新鲜制备的原代小鼠胚胎成纤维细胞作为饲养层对ES细胞的生长有明显的促进作用。     

无血清无饲养层培养体系在绵羊胚胎干细胞分离中的应用

目的：本文采用N2B27无血清无饲养层的完全已知成份的培养体系,通过机械分离法分离不同阶段的绵羊囊胚,观察不同阶段囊胚对分离绵羊类胚胎干细胞的影响。方法：本实验采用N2B27无血清无饲养层成份完全已知的培养体系,利用机械分离法对不同阶段的绵羊囊胚进行分离,观察其绵羊类胚胎干细胞的原代集落形成率,以及AKP 染色,多潜能性候选基因Oct-4 和Sox-2 免疫荧光检测。结果：分离早期阶段绵羊囊胚获得的绵羊类胚胎干细胞的形成率显著低于扩张（孵化）阶段囊胚（19.6%（11/56） vs 36.9%（31/84））（P ＜ 0.05）,同时早期和扩张（孵化）阶段绵羊囊胚的AKP 染色和多潜能性候选基因Oct-4、Sox-2 的表达呈阳性。结论：N2B27无血清无饲养层培养体系是一种有效分离绵羊类胚胎干细胞的培养基,同时分离绵羊扩张（孵化）阶段的囊胚可以显著的提高原代类胚胎干细胞的建系率,为提高绵羊类胚胎干细胞的建系奠定了基础。     

昆明小鼠胚胎成纤维细胞的分离与培养

目的:建立稳定高效的小鼠胚胎成纤维细胞饲养层培养体系.方法:取不同胎龄的小鼠分离原代胚胎成纤维细胞,观察不同胎龄小鼠对分离和培养效果的影响.结果:从不同胎龄小鼠均分离得到胚胎成纤维细胞,但最佳分离时间为13.5～14.5 d;传代时在室温下消化单层贴壁细胞可随时控制消化时间,效果良好.结论:从13.5～15.5 d胎龄小鼠胚胎分离培养胚胎成纤维细胞效果最佳.     

鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。     

昆明小鼠胚胎干细胞的体外分离培养

利用昆明小鼠13.5dpc的胚胎制备小鼠胚胎成纤维细胞,并以小鼠胚胎成纤维细胞作为饲养层;收集3.5dICR小鼠的囊胚和桑椹胚进行体外培养,筛选纯化ES细胞集落,使其稳定传代后,对其形态学和生物学性状进行初步鉴定,获得阳性细胞集落。     

国产丝裂霉素处理的胎儿成纤维细胞支持具有生殖系转化能力的小鼠胚胎干细胞的分离

应用于胚胎干细胞（embryonic stem cells, ES细胞）培养的国产药物可以降低ES细胞的实验成本。研究中以浙江海正药业生产的丝裂霉素（mitomycin，国药准字H33020786）处理小鼠胎儿成纤维细胞，用于胚胎干细胞建系。并制备饲养层，将小鼠囊胚种植在该饲养层上。4—6天后，挑选形态良好的 内细胞团(inner cell mass)来源的克隆在胰酶中进行消化，将消化下来的细胞团块传至新鲜的饲养层上。之后，每2—3天传代一次。结果表明，经该丝裂霉素处理的胎儿成纤维细胞支持具有生殖系嵌合能力的胚胎干细胞的分离。     

无血清无饲养层培养体系在绵羊胚胎干细胞分离中的应用

目的:本文采用N2B27无血清无饲养层的完全已知成份的培养体系,通过机械分离法分离不同阶段的绵羊囊胚,观察不同阶段囊胚对分离绵羊类胚胎干细胞的影响。方法:本实验采用N2B27无血清无饲养层成份完全已知的培养体系,利用机械分离法对不同阶段的绵羊囊胚进行分离,观察其绵羊类胚胎干细胞的原代集落形成率,以及AKP染色,多潜能性候选基因Oct-4和Sox-2免疫荧光检测。结果:分离早期阶段绵羊囊胚获得的绵羊类胚胎干细胞的形成率显著低于扩张(孵化)阶段囊胚(19.6%(11/56)vs 36.9%(31/84))(P0.05),同时早期和扩张(孵化)阶段绵羊囊胚的AKP染色和多潜能性候选基因Oct-4、Sox-2的表达呈阳性。结论:N2B27无血清无饲养层培养体系是一种有效分离绵羊类胚胎干细胞的培养基,同时分离绵羊扩张(孵化)阶段的囊胚可以显著的提高原代类胚胎干细胞的建系率,为提高绵羊类胚胎干细胞的建系奠定了基础。     

分离和克隆小鼠ES细胞集落的主要影响因素

目的　研究不同培养条件分离和克隆小鼠ES细胞集落的效率。方法　以PMEF饲养层、NIH3T3细胞饲养层或培养液中加入LIF为培养条件 ,分离和克隆昆明小鼠ES细胞集落 ,比较其效率。结果　饲养层的培养条件明显优于培养液中加入LIF的培养条件 ;有饲养层的培养条件下 ,桑椹胚的ES细胞集落出现率显著低于囊胚 ;两种饲养层培养囊胚 ,其ES细胞集落的出现率差异无显著性。结论　以PMEF或NIH3T3细胞作饲养层 ,培养昆明小鼠的囊胚 ,适时离散ICM ,是比较理想的分离ES细胞集落的方法。     

615小鼠ES细胞集落的分离及初步鉴定

目的　分离和培养 6 15小鼠的ES细胞集落 ,为建系打下基础。方法　以PMEF为饲养层分离 6 15小鼠的ES细胞集落 ,进行无饲养层培养 ,并对其进行初步鉴定。结果　ES细胞集落的出现率和传代成功率为 2 2 6 %和 0 94 % ,其ALP染色阳性 ,具有稳定的二倍体核型 ,可自发分化为多种类型的细胞。结论　成功分离和培养了6 15小鼠的ES细胞集落     

Isolation and culture of embryonic stem cells from porcine blastocysts

This study was conducted to establish embryonic stem (ES) cell lines from porcine blastocysts. Blastocysts were collected from China miniature pigs at day 7-9 of pregnancy. Embryos were either directly (intact embryos) cultured on mitomysin C-inactivated murine embryonic fibroblasts (MEF) as feeder layers, or were used to isolate the inner cell masses (ICM) by enzyme digestive method and then cultured. It was found that enzyme digestive method could isolate ICMs without any damages of cells in all blastocysts (28). All ICMs attached to the feeder layers. Primary cell colonies were formed in 68% of ICM culture and 28% of intact blastocyst culture. Two ES cell lines derived from ICM passed six subcultures (passages). These cells morphologically resembled mouse ES cells and consistently expressed alkaline phosphatase activity. When the ES cells were cultured in a medium without feeder layer and leukemin inhibitory factor, they differentiated into several types of cells including neuron-like, smooth muscle-like, and epithelium-like cells. Some cells formed embryoid bodies in a suspension culture. These results indicate that porcine ES cell line can be established under the present experimental conditions and these ES cells are pluripotent.     

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.     

Initial culture behaviour of rat blastocysts on selected feeder cell lines

To increase our understanding of rat embryos in culture and to attempt the isolation of blastocyst-derived cell lines, we examinated the initial growth behaviour of rat blastocysts from four strains of rat on four different feeder cell layers. The feeders used were a continuous cell line of murine embryonic fibroblasts (STO), primary mouse (MEF) or primary rat (REF) embryonic fibroblasts, and a continuous cell line of rat uterine epithelial cells (RUCs). A medium that gave optimum plating efficiencies for murine ES cells was used in the rat embryo culture. Each culture system allowed hatching and attachment of the blastocysts, that is, the behaviour was similar on each feeder and each strain for the first 2 days in culture. Subsequently, there was a rapid differentiation of the Inner Cell Mass (ICM) cells on fibroblastic feeder cell layers (STO > MEF > REF), and this was generally complete after 3–6 days in primary culture. On RUCs, the ICM was found to increase in size without differentiation up to and including day 4 and in some cases longer. Embryo-derived cells were obtained by disaggregating and passaging ICMs on REF and RUC feeders. Rounded, refractile, and epithelial-like cells were isolated on REF and colonies of ES-like cells on the RUCs. The ES-like cells were positive for expression of alkaline phosphatase and stage-specific embryonic-antigen 1. This is an important first step towards the derivation and culture of pluripotent ES cells from the rat. © 1995 Wiley-Liss, Inc.     

改进的培养体系在小鼠体细胞核移植及重构胚ES细胞分离培养中的应用

取8周后的雌性昆明小鼠进行超排,取卵母细胞用作核受体,收集卵母细胞周围的卵丘细胞作核供体,进行体细胞核移植。核移植重构胚经SrCl2激活处理6h后,与改良的M16培养液和小鼠输卵管上皮细胞共培养;将发育到早期囊胚阶段的重构胚转移至小鼠胎儿成纤维细胞饲养层上,添加含心肌细胞培养液的ES细胞培养液;把孵出的ICM进行消化接种培养,对孵出的ES细胞集落进行鉴定培养。结果显示,以小鼠卵丘细胞为核供体,体细胞核移植重构胚激活率为65．23％,囊胚发育率为11．69％;9个核移植重构囊胚中分离出ES细胞集落,分离率为2．77％;分离出的核移植ES细胞集落具有岛屿状团状隆起结构、碱性磷酸酶染色呈阳性,体外分化可形成类胚体,并能分化成上皮样或梭形细胞。ES细胞集落经常规冻存和复苏后,显示出同冻存前相似的集落形态,并具有较强的增殖能力。实验证实小鼠输卵管上皮细胞、改良的M16培养液及含心肌细胞培养液的ES细胞培养液可以更为成功地运用于小鼠的体细胞核移植及ES细胞的分离培养研究。     

Isolation and characterization of embryonic stem cell-like cells from in vitro produced goat (Capra hircus) embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P?相似文献   

Effects of feeder layer and BRL conditioned medium on mouse embryonic stem cells

In vitro growth and maintenance of embryonic stem (ES) cell lines derived from ICM cells of various blastocysts of 129 strain mice,the sustenance of their pluripotency and normal karyotype depend on the feeder layer of mouse embryonic fibroblasts (MEF).Compared with the feeder layer of MEF cells,medium conditioned by Buffalo rat liver cells (BRL-CM) is able to maintain pluripotency and karyotypic normality of ES cells only in short term cell propagation.Besides,ES cells grown in BRL-CM are also capable of aggregation with 8-cell embryos of Swiss strain and develop into germ line chimaeras.Modification to the method of aggregating ES cells with early embryos by making a hole in agar layer on the top of MEF feeder cells was shown to be more converient and efficient than the conventional microdrop method.     

Isolation and Characterization of Embryonic Stem Cell-Like Cells From in vitro Produced Goat (Capra hircus) Embryos

The aim of the present study was to isolate and characterize goat embryonic stem cell-like cells from in vitro produced goat embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 150 blastocysts and 35 hatched blastocysts whereas 100 morulae were used for blastomeres isolation mechanically. The ICM derived cells or blastomeres were cultured on a feeder layer. The primary colony formation was significantly higher (P < 0.01) for hatched blastocysts (77.14%) than early/expanded blastocysts (54%) or morula (14%). When ICMs were isolated mechanically the primary colony formation for hatched blastocysts (90%) as well as blastocysts (66%) were significantly more than when ICMs were isolated by enzymatic digestion (60% and 30%, respectively). The colonies were disaggregated either mechanically or by enzymatic digestion for further subculture. When mechanical method was followed, the colonies remained undifferentiated up to 15 passages and three ES cell-like cell lines were produced (gES-1, gES-2, and gES-3). However, enzymatic disaggregation resulted in differentiation. The undifferentiated cells showed stem cell like morphological features, normal karyotype, and expressed stem cell specific surface markers like alkaline phosphatase, TRA-1-61, TRA-1-81, and intracellular markers Oct4, Sox2, and Nanog. Following prolonged culture of the ES cell-like cells were differentiated into several types of cells including neuron like and epithelium-like cells. In conclusion, goat embryonic stem cell-like cells can be isolated from in vitro produced goat embryos and can be maintained for long periods in culture.