共查询到16条相似文献，搜索用时 265 毫秒
1.
小鼠皮肤成纤维细胞的体细胞核移植 总被引：1，自引：1，他引：0
取成年小鼠唇部皮肤进行培养，分离成纤维细胞并血清饥饿培养1周，用作核供体。对成年小鼠进行超排，取卵母细胞用作核受体，核移植重构胚经SrCl2激活处理6h后，同mM16培养液和小鼠输卵管上皮细胞共培养，把发育到早期囊胚的重构胚转移至小鼠胎儿成纤维细胞饲养层上，添加ES细胞条件培养液，消化分离ICM，然后接种培养，对孵出的ES细胞样集落进行鉴定培养。结果显示，小鼠唇部皮肤成纤维细胞为核供体，核移植重构胚2-细胞率为54．05％，桑椹胚率17．14％，囊胚率6．90％，对照组卵丘细胞的核移植重构胚2-细胞率为60．00％，桑椹胚率21．85％，囊胚率11．69％，但2种供体细胞在支持核移植重构胚发育能力上差异不显著。成纤维细胞重构囊胚中6个囊胚分离出ES细胞样集落，3个ES细胞样集落可稳定传代；对照组卵丘细胞重构囊胚中9个囊胚中分离出ES细胞样集落，5个ES细胞样集落可稳定传代。从核移植重构胚中分离出的ES细胞样集落具有岛状或巢状群体生长形态，生长旺盛的集落可自发分化成单个散在或片状存在的上皮样或梭形细胞，碱性磷酸酶检测为阳性，常规冻存复苏，仍显示ES细胞特征。 相似文献
2.
以昆明系小鼠为对象,经过丝裂霉C处理成纤维细胞(Mouse embryonic fibroblast,MEF)制备饲养层,对影响小鼠胚胎干细胞(Embryonic stem cell,ES细胞)分离培养的相关因素进行研究。分别收集小鼠3.5d的囊胚(扩张囊胚)和4.5d囊胚(孵化囊胚)进行培养,比较扩张囊胚和孵化囊胚的贴壁率、原代克隆率及传代率的情况。收集3.5d胚龄的囊胚,通过全胚法和免疫外科法对内细胞团(Inner cell mass,ICM)进行分离培养ICM集落,确定离散ICM的适宜时间。用0.25%胰酶+0.04%EDTA,0.125%胰酶+0.02%EDTA和0.25%胰酶+1%小鸡血清等方法对小鼠ES细胞集落进行传代,观察不同酶浓度对ES细胞分离克隆的影响。结果显示,孵化囊胚的贴壁率高于扩张囊胚(P0.05),但传代率则相反(P0.05),原代克隆率差异不显著(P0.05);一般ICM增殖培养2～3d(免疫外科法)或4～5d(全胚培养法)后,出现典型的克隆集落,再挑取ICM;0.125%胰酶+0.02%EDTA及0.25%胰酶+1%小鸡血清,形成ES原代克隆率较高,2组没有显著性差异(P0.05);结果表明,分离得到的ES细胞经形态学观察,AKP染色,体外分化试验等表明其具有胚胎干细胞的特性。 相似文献
3.
ICR小鼠胚胎干细胞建系初步研究 总被引：1，自引：0，他引：1
实验旨在探讨消化方式和胚胎发育阶段对ICR小鼠胚胎干细胞(ES细胞)建系效率的影响。ICR小鼠3.5 d囊胚在饲养层上贴壁后采用单一酶消化或机械化与酶消化法相结合分离隆起的细胞集落,进行传代培养;然后选择二者中较优消化方式对不同发育时期囊胚所形成的细胞集落进行处理。结果表明:采用机械化与胰酶消化相结合的方式,形成的类ES细胞超过7代的比率(85.0%)要显著高于单一的胰酶消化(15.0%)(P<0.05);当用二者相结合的方式对ICR小鼠3.5 d(早期囊胚)、4.0 d(扩张囊胚)和4.5 d(孵化囊胚)所形成的细胞集落进行消化传代培养,三者在贴壁率和形成原代细胞集落率上均无显著差别(P>0.05),但传代超过7代的效率上早期囊胚和扩张囊胚均高于孵化囊胚(P<0.05)。结果提示,采用机械化与酶消化法相结合更适合于3.5～4.0 d ICR小鼠囊胚的ES细胞建系。 相似文献
4.
山羊类ES细胞的分离与克隆 总被引：6，自引：0，他引：6
采集山羊交配后6～8d的桑椹胚、囊胚和孵化囊胚,将桑椹胚和囊胚分别放在小鼠原代胎儿成纤维细胞(PMEF)饲养层和同源原代胎儿成纤维细胞(PGEF)饲养层上比较其脱带时间及脱带率。脱带后,将各自一半胚胎切割,把含ICM的半胚分别放在相应饲养层上进行培养,另一半整胚在各自饲养层上继续培养,而孵化囊胚直接于PGEF饲养层上培养。当ICM增殖一定程度时进行传代,以比较其类ES细胞分离与克隆的效果。结果表明,在2种不同饲养层上,囊胚的脱带时间均短于桑椹胚,囊胚的脱带率均高于桑椹胚,而饲养层的种类对胚胎的脱带时间以及脱带率影响不大。脱带切割囊胚不论在PMEF还是在PGEF饲养层上,其贴壁时间均短于脱带整胚及孵化囊胚,而贴壁率高于脱带整胚,与孵化囊胚相似。脱带整胚及脱带切割胚在PMEF饲养层上所获类ES细胞只能维持3代,而在PGEF饲养层上,脱带切割半胚和孵化囊胚所获类ES细胞传至5代。由此认为,对脱带后的胚胎进行切割处理,有利于ICM的贴壁和增殖;应用同源原代胎儿成纤维细胞饲养层培养系统,有利于类ES细胞的分离与克隆。 相似文献
5.
为了更高效地分离昆明小鼠胚胎干细胞,本研究从饲养层、胚胎发育阶段和培养液方面进行优化。将3代以内的小鼠胎儿成纤维细胞(MEF)用丝裂霉素C处理后,分别按1×104、1×105、1×106·mL-1密度接种,以H-DMEM+15%KSR+LIF为培养液,观察不同密度饲养层对昆明小鼠胚胎干细胞(ES细胞)生长的影响,并研究胚胎发育阶段和培养液中分别添加干细胞生长因子(SCF)、SCF+胰岛素对昆明小鼠ES细胞分离克隆的影响。结果显示,胚胎在密度为1×105·mL-1的饲养层上,F1代和F2代ES细胞克隆形成率均显著高于其他2组(P<0.05)。囊胚的F2代ES细胞克隆形成率显著高于桑椹胚(P<0.05),培养液中添加SCF显著提高昆明小鼠胚胎贴壁率(P<0.05),同时添加SCF和胰岛素得到昆明小鼠最高胚胎贴壁率及F1、F2代ES细胞克隆形成率。所分离的ES细胞显示AKP染色强阳性,Oct-4、SSEA-1的免疫荧光检测阳性,具有ES细胞的特点。由此认为,发育至囊胚的胚胎在MEF密度为1×105·mL-1上,培养液中同时添加SCF和胰岛素更适合昆明小鼠ES细胞的分离培养。 相似文献
6.
本研究旨在筛选获取优质ICM集落的实验方法。以昆明系小鼠为实验动物,采集3.5、4 d和4.5 d的胚胎,分别移入DMEM培养液、DMEM+白血病抑制因子(LIF)和小鼠胎儿成纤维细胞共培养体系中进行培养,观察比较内细胞团(ICM)从透明带中孵出的时间、孵化囊胚贴壁情况以及ICM集落形成情况。结果表明:妊娠3.5 d的胚胎61%为桑椹胚,需要经过4～5 d的体外培养才能形成ICM集落;妊娠4 d的扩张囊胚经过3～4 d的共培养后形成ICM集落;妊娠4.5 d的孵化囊胚经过1～2 d的共培养即可形成ICM集落;ICM形成率以妊娠4.5 d的胚胎最高,显著高于妊娠4 d和3.5 d的胚胎(P<0.01),但妊娠4.5 d冲出孵化囊胚数量显著降低(P<0.01);小鼠胎儿成纤维细胞共培养体系优于DMEM和DMEM+LIF培养液。因此,采集妊娠4 d的昆明小鼠胚胎,选择小鼠胎儿成纤维细胞共培养体系,在体外培养3～4 d,能够有效分离培养出可用于胚胎干细胞研究的优质ICM。 相似文献
7.
将187枚山羊胚胎按如下方式培养在GEF饲养层上:①桑椹胚直接培养;②囊胚直接培养;③囊胚去透明带后培养;④囊胚去透明带并被撕裂成碎片后培养;⑤囊胚去透明带后胰蛋白酶消化成小细胞团培养。将上述培养所获取的18个内细胞团(ICM)均分为2组,第1组始终从生长物中挑取较为浓密的类ES细胞团传代,第2组始终让培养物生长到铺满培养皿(瓶)底再传代。结果显示:①桑椹胚、囊胚直接培养以及囊胚被消化成小细胞团后培养,均未获得ICM;囊胚去透明带后培养,获得ICM的数量占参试囊胚数的30%;囊胚去透明带并被撕裂成碎片后培养,获得ICM的数量占参试囊胚数的60%。②始终从培养物中挑取类ES细胞团进行传代,传至第4代后已无细胞可传;而将培养物培养至长满培养皿(瓶)底后再一起传代,细胞可传11代以上,且其中的类ES细胞团呈大量扩增趋势。 相似文献
8.
本实验旨在探讨昆明白小鼠桑椹胚超低温冷冻后Oct-4表达的变化与胚胎发育潜力的关系。胚胎采用OPS法冷冻,即胚胎于10%EG+10%DMSO溶液中预处理30 s,然后再移入玻璃化溶液(EDFS30)中处理25 s,以OPS为承载器投入液氮中。毒性组胚胎未投入液氮,其他过程与冷冻组相同,新鲜胚胎为对照组。胚胎解冻后,检测Oct-4 mRNA与蛋白的表达,通过观察其形态正常率、囊胚发育率和囊胚细胞数来综合判断其体外发育能力。结果表明:冷冻组和毒性组较桑椹胚中Oct-4 mRNA的表达量对照组相比显著降低(P<0.05),蛋白表达量3组间无显著差异。桑椹胚处理后的形态正常率以及桑椹胚培养48 h后的囊胚发育率(96.67%～100%)和囊胚细胞数(89.67～92.33)3组间无显著差异。结果显示,玻璃化冷冻保存小鼠桑椹胚后多能性基因Oct-4 mRNA表达的变化并未影响其体外发育潜力。 相似文献
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建立有效的昆明白小鼠胎儿成纤维细胞(Mouse embryonic fibroblast,MEF)饲养层培养体系,用于分离和培养小鼠胚胎干细胞(Embryonic stem cell,ES细胞)的研究.(1)取怀孕14.5 d昆明白小鼠胎儿分离成纤维细胞,利用体外培养体系分离传代,选取生长旺盛并且已纯化的P3代的MEF,经丝裂霉素处理后.用细胞计数板计算活细胞数,分别按1×104、1×106、1×108/mL密度接种,制备饲养层,观察不同密度饲养层的生长状况.(2)取怀孕4 d的昆明白小鼠囊胚,接种在不同密度饲养层上.观察不同密度饲养层上囊胚、ICM及ES细胞的克隆生长情况.结果显示:囊胚在密度为1 × 106/mL的饲养层上,贴壁率和ICM孵出率分别为(97.0±3.606)%和(96.3±2.887)%,显著高于其他2组;密度为1×104/mL饲养层上的ES细胞克隆形成率高于密度为1×104/mL(差异极显著,P<0.01)和1×108/mL饲养层上的ES细胞克隆形成率(差异显著,P<0.05);而1×104/mL饲养层和1×108/mL饲养层上的ES细胞克隆形成率差异不显著(P>0.05).结果表明:以1×104/mL密度接种的MEF作为饲养层,最适合用于分离培养昆明白小鼠ES细胞,有利于囊胚的发育,ICM的增殖,促进ES细胞的增殖,并起到抑制其分化的作用. 相似文献
10.
《畜牧兽医学报》2015,(10)
旨在为小鼠和猪多能性干细胞嵌合体制作提供参考,探讨胚胎干细胞(Embryonic stem cells或Embryonic germ cells,ES/EG)显微注射及受体胚胎发育时期等对胚胎发育的影响。对小鼠ES细胞和猪EG细胞分别进行研究:(1)将小鼠ES细胞分别注入小鼠桑椹胚和囊胚,比较不同发育时期受体胚胎体外培养的发育率和孵化率,以及注射胚胎和未注射胚胎体外培养的发育率和孵化率。结果表明,显微注射后的桑椹胚体外培养发育率和孵化率分别为94.7%和70.2%,囊胚率分别为84.6%和80.8%,注射后桑椹胚体外培养发育率极显著高于囊胚(P0.01),但注射后囊胚体外培养孵化率极显著高于桑椹胚(P0.01);未注射胚胎(注射胚胎)的体外培养发育率和孵化率分别为96.6%(89.9%)和67.8%(75.2%),未注射胚胎发育率极显著高于注射胚胎(P0.01),但注射胚胎孵化率极显著高于未注射胚胎(P0.01)。(2)将猪EG细胞分别注入猪桑椹胚、早囊、囊胚及孵化囊胚,比较不同发育时期受体胚胎移植后妊娠率以及注射胚胎和未注射胚胎体外培养的孵化率,并对猪EG细胞显微注射针和固定针的规格进行了探讨。结果表明,显微注射的桑椹胚、早囊及囊胚移植后妊娠率为67%,而显微注射的孵化囊胚移植妊娠率为0,桑椹胚、早囊及囊胚移植后受体母猪妊娠率极显著高于孵化囊胚(P0.01),由桑椹胚、早囊及囊胚注射后移植获得了两头嵌合体仔猪;显微注射和未显微注射的猪胚胎其孵化率分别为47.54%和72.97%,未注射胚胎孵化率极显著高于注射胚胎(P0.01)。确定猪EG嵌合体制作过程中,注射针外径约为30μm,内径约为25μm;固定针外直径约为130~150μm,内径约为40μm。嵌合体制作时,胚龄宜早不宜迟,桑椹胚显微注射更易操作,而且导入的时期较早,ES/EG细胞在嵌合体组织中可以有很高的贡献率,故选择桑椹胚进行注射。在本试验条件下,显微注射对小鼠胚胎发育影响不大,对猪胚胎影响较大,说明猪胚胎显微注射条件需要进一步优化。 相似文献
11.
以猪孤雌激活囊胚为材料,囊胚透明带消化后采用全胚培养,培养液中添加不同培养成分或因子（如FGF2,LIF,2i等）,以及选择不同的初始培养液体积来筛选猪胚胎干细胞（embryonic stem cell,ES细胞）建系的优化培养体系。囊胚内细胞团形成的细胞集落采用胰酶消化传代。结果显示：透明带消化后,囊胚贴壁率显著升高（19．4％VS．8．8％）（P〈0．05）;初始培养液体积比平常培养液体积（0．30mL／孔,24孔培养板）减半条件下,能显著提高其贴壁率（91．7％VS 20．0％）（P〈0．01）,而且获得了可传至7代的类ES细胞系2株,碱性磷酸酶染色成阳性;当用2i因子（CHIR99021和PD03025901）去替代培养液中的FGF2,囊胚贴壁率（29．400VS53．3％）和原代集落形成率（20．0％VS 87．5％）反而显著下降（P〈0．01）。这表明培养液添加了FGF2和LIF（不舍2i因子）,用24孔板培养,最初培养体积为0．15mL,透明带消化的培养体系比较适合猪孤雌激活胚的ES细胞建系。 相似文献
12.
Li M Ma W Hou Y Sun XF Sun QY Wang WH 《The Journal of reproduction and development》2004,50(2):237-244
Pigs serve as a better research model for human beings than other species. The Chinese laboratory miniature pig is a new laboratory animal and is expected to be applicable in many medical research fields. This study was to establish effective technologies to isolate and culture ES cells in Chinese miniature pigs. For isolation of the inner cell mass from blastocysts, an enzyme-digestive method was compared with the traditional immunosurgery. Isolated ICM were cultured in three feeder cell layers: mouse embryonic fibroblasts (MEF), porcine embryonic fibroblasts (PEF) and a continuous cell line of mouse embryonic fibroblasts (STO). Microtubule activity of the three feeder cells was further examined by immunofluorescence. ICM were successfully isolated from 85% of blastocysts by the enzyme-digestive method, compared to only 40% by immunosurgery. When ICM were cultured in three feeder layers for two to three days, 75%, 65% and 20% of ICMs formed primary cell colonies in MEF, PEF and STO, respectively. Colonies were also formed during subcultures after 9, 5 and 1 passage in MEF, PEF and STO, respectively. Microtubules in STO cells were significantly fewer than those in MEF and PEF. When the ES-like cells were cultured in a differentiation medium, they differentiated to neuron-like cells and other types of cells. These results indicate that healthier ICM can be obtained with the enzyme-digestive method. Successful culture of ICM to ES-like cells has been achieved not only in MEF, but also in homologous (pig) feeder layer. The ES cells obtained in the present study were pluripotent. 相似文献
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14.
Oliveira CS de Souza MM Saraiva NZ Tetzner TA Lima MR Lopes FL Garcia JM 《Reproduction in domestic animals》2012,47(3):428-435
Despite extensive efforts, establishment of bovine embryonic stem (ES) cell lines has not been successful. We hypothesized that culture conditions for in vitro-produced (IVP) embryos, the most used source of inner cell mass (ICM) to obtain ES cells, might affect their undifferentiated state. Therefore, the aim of this work was to improve pluripotency of IVP blastocysts to produce suitable ICM for further culturing. We tested KSR and foetal calf serum (FCS) supplements in SOF medium and ES cell conditioned medium (CM) on IVC (groups: KSR, KSR CM, FCS and FCS CM). Cleavage and blastocyst rates were similar between all groups. Also, embryonic quality, assessed by apoptosis rates (TUNEL assay), total cell number and ICM percentage did not differ between experimental groups. However, expression of pluripotency-related markers was affected. We detected down-regulation of OCT3/4, SOX2 and SSEA1 in ICM of FCS CM blastocysts (p < 0.05). SOX2 gene expression revealed lower levels (p < 0.05) on KSR CM blastocysts and a remarkable variation in SOX2 mRNA levels on FCS-supplemented blastocysts. In conclusion, pluripotency-related markers tend to decrease after supplementation with ES cell CM, suggesting different mechanisms regulating mouse and bovine pluripotency. KSR supplementation did not differ from FCS, but FCS replacement by KSR may produce blastocysts with stable SOX2 gene expression levels. 相似文献
15.
The objective of this study was to use mouse embryos as a model system to investigate the effect of co-culture of cumulus cells in Sydney IVF sequential media (Cook) on embryo development, based on the hypothesis that feeder cells in co-culture with a sequential medium could work synergistically to further improve in vitro culture conditions for mammalian preimplantation embryos. The culture systems described here were evaluated by the ability to consistently produce high blastocyst formation rates and high cell number per blastocyst. The role of embryo-to-cell contact was assessed by using Transwell inserts with transparent microporous membranes. Pronuclear embryos of ICR mice were cultured to blastocysts in Cook sequential media, with and without mouse primary cultures of cumulus cells, and with or without inserts. Blastocyst formation rates and cell numbers of in vitro developing embryos in the different culture systems were compared to each other, and to in vivo derived blastocysts. Blastocyst formation rates for Cook medium only was 27.8% (without inserts) and 32.9% (with inserts), whereas Cook-Cumulus cells in identical culture systems was significantly higher at 45.8% (without inserts, P<0.05) and 55.6% (with inserts, P<0.05). When the embryos are suspended above the bottom of the well, for Cook medium significantly lower blastocyst formation rates were observed at 4.2% compared to Cook-Cumulus cells at 17.5% (P<0.05). Mean cell numbers of blastocysts obtained in all co-culture systems were significantly higher (P<0.05) compared to those developing in culture medium only. Although the putative mechanism is as yet unexplained, the improved blastocyst formation rates and cell numbers in co-culture when there is direct contact between the embryo and the cell monolayer suggest that the close proximity between the feeder cells and embryos is in part responsible for these effects. 相似文献
16.
对影响小鼠胚胎干细胞（Embryonic stem cells,ES细胞）培养、克隆、分离、传代效果的因素进行了探索研究。应用223枚昆明白小鼠胚胎和20枚129品系小鼠胚胎的研究结果表明，129品系小鼠胚胎比昆明白小鼠胚胎更适合作为ES细胞建系的材料，两者FS出现率差异显著（P＜0．05）；以DMEM＋10％NBS＋10％FCS为基础培养液，分别加入LIF、胰岛素、LIF＋SCF，极显著提高昆明白小鼠胚胎贴壁率，ICM生长率及F1、F2出现率（P＜0．01），而在DMEM＋10％NBS＋10％FCS＋LIF＋SCF为培养液，得到昆明白小鼠胚胎最高贴壁率、ICM生长率及传代率；4dpc胚胎传代情况显著好于3．5dpc胚胎（P＜0．05）。 相似文献