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从胚胎发育阶段、饲养层和培养体系等方面对影响绵羊类ES细胞分离、克隆效率的因素进行探讨。结果显示:致密桑葚胚和囊胚的ICM增殖率高于囊胚和孵化囊胚。绵羊类ES细胞在同源绵羊胎儿成纤维细胞(SEF)上生长比较缓慢,最终传代次数也低于小鼠胎儿成纤维细胞(MEF)组。培养液中同时添加胎牛血清(FBS)和Knock-out血清替代品(KSR),绵羊类ES传至7代,添加了碱性成纤维细胞生长因子(bFGF)后,最高可传至8代,而单纯添加KSR或FBS,分别传至4代和5代。对类ES细胞进行AKP染色、核型分析、体外分化试验,证实分离的类ES细胞符合ES细胞的主要特征,而且表达多潜能性细胞因子Nanog。由此认为,致密桑葚胚和囊胚更适合绵羊类ES细胞的体外分离和培养,而且MEF更适合于绵羊类ES细胞的分离传代,培养液中添加5%FBS和15%KSR,比较适合类ES细胞的分离传代,bFGF对绵羊类ES细胞的增殖具有促进作用。 相似文献
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将本地槐山羊胎儿的原始生殖细胞(PGCs)与其生殖嵴周围组织细胞共同分离,经传代培养后获得了具有干细胞特征的山羊类ES细胞。结果表明高糖DMEM培养基和低糖DMEM培养基相比较,低糖DMEM更适宜于山羊类ES细胞的分离与克隆;类ES细胞在山羊胎儿成纤维细胞饲养层上生长效果较好,可传4代或5代,而在小鼠成纤维细胞饲养层上,类ES细胞仅传3代;联合添加白血病抑制因子(LIF)、干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)能显著提高山羊类ES细胞分离与克隆的效率;胎龄为30~45d的胎儿原代培养时可获得大量的细胞集落,克隆培养可传至5代,适合作山羊类ES细胞的分离培养。 相似文献
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山羊PGCs用于分离与克隆类ES细胞 总被引:11,自引:1,他引:10
选择健康成年本地白山羊,自然发情,配种后44d取胎儿,以传统的原始生殖细胞(PGCs)分离与克隆的方法和PGCs与其胎儿生殖嵴周围组织细胞共同培养的方法获得类胚胎干细胞(类ES细胞),并对山羊类ES细胞在不同饲养层上进行培养。结果表明,采用传统方法与共培养的方法并添加细胞因子均能分离获得类ES细胞。分离获得的类ES细胞在同源(山羊)胎儿细胞饲养层上生长效果较好,可传4代或5代,而在小鼠原代成纤维细胞饲养层上类ES细胞仅传3代。另外,共培养不添加细胞因子组仅获1个ES细胞集落,传代后丢失。 相似文献
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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
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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
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旨在优化并建立有利于山羊类ES细胞生长的饲养层细胞、培养基、细胞因子和传代方法。本研究选择武汉市本地白山羊配种6~7 d后的胚胎,通过全胚培养法、酶消化与机械分离结合法分离山羊类ES细胞,并在不同的细胞饲养层中饲养、在培养基中添加不同的生长因子、采用不同的传代方法,比较不同培养条件对山羊类ES细胞生长和增殖的影响。通过碱性磷酸酶染色和免疫组化染色法鉴定ES细胞特异生物标记AKT、SSEA-1和Oct-4的表达,并将得到的山羊类ES细胞进行体外分化。结果表明,与小鼠胎儿成纤维细胞(Mouse embryonic fibro-blast,MEF)和山羊胎儿成纤维细胞(Goat embryonic fibroblast,GEF)相比,C2C12更有利于细胞的贴壁,但差异不显著(P>0.05);细胞传代时,在高浓度细胞因子LIF(Leukemia inhibitory factor)和SCF(Stemcell factor)中处理ICM(Inner cell mass),更有利于细胞传代(传至第8代);培养基中添加LIF和SCF的同时,添加肝素和胰岛素,更有利于细胞的传代(传至第9代);山羊类ES细胞中SSEA-1(... 相似文献
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比较了在无血清培养体系中分别添加小分子化学物质pluripotin(SCl)和白血病抑制因子(LIF)对昆明小鼠ES细胞未分化状态的维持作用,并探讨了SC1在培养液中的最佳浓度.以小鼠胎儿成纤维细胞(MEF)为饲养层,以含LIF的添加血清替代品(KSR)的培养基(ESC-SR)为对照,分别用SC1浓度为0.3、1、3μmol/L的ESC-SR培养液对昆明小鼠胚胎进行分离培养.结果显示:胚胎在添加3 μmol/L SC1的ESC-SR培养液中ICM形成率显著高于对照组(P<0.05),形成的ES集落与对照组形态差异不明显,最高传至第7代,并且在无饲养层条件下可以传至第6代而保持未分化状态.不同浓度SC1培养液中,胚胎在SC1浓度为0.3 μmol/L组的F2代形成率显著高于1 μmol/L组和3μmol/L组(P<0.05),所分离的细胞AKP染色呈阳性,Oct4、Nanog和Sox2的免疫荧光染色均显阳性,具有ES细胞的特点.结果表明,ESC-SR培养液中SC1可以替代LIF用于小鼠ES细胞未分化状态的维持,并且可以在无饲养层条件下进行昆明小鼠ES细胞的培养. 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
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魏静 《四川畜牧兽医学院学报》2009,(4):28-32
在现代法律秩序中,商会自治规范是制定法的基础和必要的补充,甚至在某些方面替代了制定法;商会自治规范主要包括商会组织规范、行为规范、惩罚规范以及争端解决规范等;其效力仅及于其内部成员;商会自治规范和制定法之间存在冲突,但也存在整合的基础。 相似文献
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本文概述了猪的毛色类型、猪的毛色遗传模式,着重综述了猪毛色基因分子基础的研究进展,指出存在问题并就未来发展方向做了思考。 相似文献
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REASONS FOR PERFORMING STUDY: Centesis of the bicipital bursa using an 8.9 cm long spinal needle has been reported but the alternative of employing a 3.8 cm long hypodermic needle requires validation. OBJECTIVE: To compare the efficacy of 2 different methods of centesis of the bicipital bursa and to evaluate the usefulness of ultrasonographic imaging to determine the location of solution administered when centesis of the bursa is attempted. METHODS: For Trial 1, 6 clinicians, who had no previous experience of centesis of the bicipital bursa, attempted to inject a solution composed of an aqueous radiopaque contrast medium and physiological saline solution (PSS) into the bicipital bursae of 2/12 horses using the previously described distal approach to inject one bursa and a proximal approach to inject the contralateral bursa. The bicipital tendon and bursa were examined ultrasonographically before and after injection; and both shoulders were examined radiographically to identify the location of the medium. In Trial 2, another 6 clinicians, also with no previous experience of centesis, repeated Trial 1, using 6 horses, but the radiopaque contrast medium was mixed with air instead of PSS. RESULTS: Accuracy of centesis using the proximal approach was 39% and that of the distal approach 28%. Ultrasonographic examination of the shoulder allowed the location of solution and air to be accurately predicted in all 12 shoulders examined. CONCLUSIONS: Clinicians who have had no previous experience performing centesis of the bicipital bursa are unlikely to be successful in centesis using either approach. Radiographic examination after injecting a radiopaque contrast medium may be necessary to assess the success of centesis especially if bursal fluid is not obtained during centesis. Injecting air along with the radiopaque contrast medium provides more accurate ultrasonographic confirmation of centesis and better radiographic definition than does injection without air. 相似文献
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以国际标准强毒R株人工感染非免疫产蛋鸡,定时扑杀,分别从鼻窦、眶下孔、气管、肺、气囊、卵巢和输卵管分离MG,并收集感染鸡所产蛋分离MG。结果表明,人工感染48小时后上、下呼吸道及肺已被全面感染,96小时气囊已被感染,120小时输卵管已能分离到MG,卵巢始终分离不到MG。人工感染鸡自144小时便能在其所产蛋中分离出MG。药物治疗能在72小时内消除感染,油乳剂苗则需24天后逐渐降低蛋内MG分离率,药物卵内注射、种蛋药浴、高温处理均能杀死卵内MG,但以研制的种蛋浸泡剂药浴效果为最好。 相似文献
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Effects of size of ingestively masticated fragments of plant tissues on kinetics of digestion of NDF
Ingestively masticated fragments were collected and sized via sieving. Different sizes of esophageal masticate and ruminal digesta fragments, and ground fragments of larger masticated pieces were incubated in vitro, and undigested NDF remaining at intervals of up to 168 h of incubation was determined. The ruminal age-dependent time delay (tau) for onset of digestion of NDF was positively correlated (P < 0.004) with the mean sieve aperture estimated to retain 50% of the fragments between successive sieve apertures (MRA). Degradation rate of potentially degradable NDF (PDF) and level of indigestible NDF were not related (P > 0.10) to MRA of masticated and ground fragments. Estimates of tau were positively related to MRA, with slopes of bermudagrass < corn silage < ruminal fragments of corn silage. It was concluded that fragment size-, and consequently, ruminal age-dependent onset of PDF degradation of a mixture of different fragment sizes results in an age-dependent rate of degradation of the more rapidly degrading of two subentities of PDF. Models are proposed that assume a tau before onset of simultaneous degradation of PDF from two pools characterized as having gamma-modeled age-dependency and age-constant rates. The ruminal age-dependent pool seems to be associated with the faster-degrading pool, and its rate parameter increases with range in MRA in the population of fragments. Conceptually, the ruminal age-dependent rate parameter for PDF degradation seems to represent a composite of several effects: 1) effects of the size-dependent tau; 2) range in MRA of the population of ingestively masticated fragments; and 3) subentities of PDF that degrade via more rapid age-dependent rates compared with subentities of PDF that degrade via age-constant rates. The estimated fractional rates of ruminative comminution of ingestively masticated fragments (0.060 to 0.075/h) were of a magnitude similar to the mean fractional rates of PDF digestion (0.030 to 0.085/h), which implies that ruminative comminution may be first-limiting to fractional rate of PDF digestion. The in vivo roles of ingestive and ruminative mastication of fragments on PDF degradation must be considered in any kinetic system for estimating PDF digestion in the rumen. These results and others in the literature suggest that the rate of surface area exposure rather than intrinsic chemical attributes of PDF may be first-limiting to degradation rate of PDF in vivo. 相似文献