牛胚胎生殖细胞的分离与培养

Isolation and Culture of Bovine Embryonic Germ Cells

以牛胎儿为材料,从原始生殖细胞(PGC)分离培养出胚胎生殖细胞(EG),并进行传代和鉴定,对影响胚胎生殖细胞分离与培养的因素进行了探讨,研究发现以共培养方式培养牛原始生殖细胞时,原代培养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地支持PGC的生长和增殖,组织块培养细胞克隆数目较少,PGC很难增殖,不能形成典型的集落,传代也不理想,只传了一代。同时比较了不同传代方法对牛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。原代培养的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成纤维样细胞、上皮样细胞和类胚体。

The major object of this paper is to isolate and culture bovine 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 ECr cells have been discussed. When co-culture the EG cells with somatic cells, there will be some colony in primary culture, which maybe account for homologous somatic cells can sustain the EG survive and proliferation. When we use the tissue culture method, the colony of EG cells is very less and EG cells cannot proliferate effectively, as well as the passage culture. With 0. 125% trypsinase ＋ 0.02% EDTA disrupt gonad, comparing effect of different dispersal methods on the passage of the 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. An Immunohistochemistry assay was carried out to characterize the colonies of bovine primory EG cells, and expression of stage-specific embryonic antigen SSEA-1, 3, 4 was detected, but the expression was feeble. The EG cells can differentiate into fibroblast-like cells, epithelia-like cells and embryoid body.