【作者】 葛秀国；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2004， 硕士

【摘要】 本研究以牛和山羊胎儿为材料，从原始生殖细胞分离培养出EG细胞，并进行传代培养和鉴定，对影响胚胎生殖细胞(Embryonic germ cell,EG)分离与培养的影响因素进行了探讨，为进一步建立牛和山羊EG细胞系奠定了基础。实验主要结果如下：从49例牛胎儿分离培养胎儿原始生殖细胞（Primordial germ cell，PGC），胎龄在29~50d的牛胎儿可以用于分离培养原始生殖细胞，最高传至7代。统计46例山羊胎儿分离培养原始生殖细胞情况，胎龄在25~38d的胎儿原代培养时都可获得大量的细胞集落，适合做EG细胞的分离培养，最高传至6代。用不同的饲养层培养牛原始生殖细胞，实验结果表明MEF，STO和BEF饲养层都能支持牛原代PGCs的增殖，但MEF较BEF和STO效果更好以MEF、GEF、BEF为饲养层，在不添加细胞因子情况下，都可以培养出原代山羊的胚胎生殖细胞。传代结果表明，MEF的培养效果较好，但与GEF组二者差异不显著(P>0.05)，BEF组培养效果较差（P<0.05）。以共培养方式培养牛、山羊原始生殖细胞，在原代培养时，都可以出现大量形态较好的EG细胞集落，说明同源的体细胞可以很好的支持PGC的生长和增殖。试验以三个不同浓度LIF的培养液培养山羊PGCs。在原代培养时，效果差异不显著(P>0.05)；而在传代过程中，以含LIF 10 ng/mL组培养效果最好，但与含LIF 5 ng/mL组差异不显著(P>0.05)，LIF 1 ng/mL组培养效果较差(P<0.05)。试验比较了不同消化方法对山羊胎儿原始生殖细胞分离的影响。在37℃下，0.125%胰蛋白酶+0.02% EDTA（处理20~30 min）消化分离效果较好于0.25%胰蛋白酶+0.04% EDTA（处理15 min）试验比较了不同传代方法对牛和山羊胚胎生殖细胞细胞传代的影响，发现消化+机械分离法和消化+吹打法都可以用于EG细胞的传代。消化+吹打法操作简单，省时省力，也能够很好的保持细胞的增殖活力。牛和山羊的胚胎生殖细胞集落碱性磷酸酶鉴定为阳性。牛原代胚胎生殖细胞进行了细胞表面标志抗原SSEA-1，3，4鉴定，呈弱阳性。细胞体外培养可以分化为脂肪细胞、成纤维样细胞、上皮样细胞和类胚体。更多还原

【Abstract】 The major object of this paper is to isolate and culture bovine and goat embryonic germ (EG) cells derived from primordial germ cells (PGCs) as well as to identify the EG cells from various aspects. Some factors influencing the efficiency of the isolation and culture bovine and goat EG cells have been discussed. Our work may be useful to further establish bovine and goat EG cell lines. The results obtained were as follows:Isolation and culture of bovine primordial germ cells was studied in 49 fetuses .We found that the bovine fetuses with the age from 29 to 50 days can be used to isolate the primordial germ cells, and one EG cell line maintained undifferentiation for 7 passages.The primordial germ cells were isolated from 46 goat fetuses embryonic age from 25 to 38 days, there were so many colonies in primary culture. One EG cell line maintained undifferentiation for 6 passages.When bovine primordial germ cells were cultured on different feeder cells: MEF，STO and BEF, all can sustain bovine primary embryonic germ cells, and MEF was the best.When goat primordial germ cells were cultured on different feeder cells, MEF，GEF and BEF all can sustain goat primary embryonic germ cells, and MEF was finer than others, but there were no significant difference between MEF and GEF（P>0.05）.When the goat primordial germ cell and somatic cell were co-cultured, there are some colonies in primary culture, because the somatic cell can maintain the survival of the PGCs.Goat primordial germ cells were cultured with the medium contained different concentration of LIF, there was no difference in primary culture (P>0.05); and when passaged, the group contain 10 ng/mL LIF was finer than others, but there was no difference with the the group contain 5 ng/mL LIF (P>0.05). The group contain 1ng/mL LIF was the worst.Another experiment was performed to compare the effect of different methods of disruption on goat EG isolation. The dispersal effect of 0.125% trypsinase + 0.02% EDTA (disposed 20~30 min) was finer than that of 0.25% trypsinase + 0.04% EDTA (disposed 15 min) at 37℃.With 0.125% trypsinase + 0.02% EDTA disrupt gonad, comparing effect of different dispersal methods on the passage of goat EG cells, the method of “digest + blowing disperse” was fine, which was simple and can save more time and labours and better to keep the activity of the EG cells. <WP=8>One identification of PGC-like cell were accomplished by alkaline phosphatase(AKP) staining, and AKP-positive cells and colonies were found. An Immunohistochemistry assay was carried out to characterize the colonies of bovine primordial germ cells, and expression of stage-specific embryonic antigen 1, 3, 4 was detected, but the expression was feeble.更多还原