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目的成功建立小鼠胚胎生殖细胞(EGCs)系,并初步分析小鼠胚胎生殖细胞的印记状态。方法建立交配后12.5d(12.5dpc)原始生殖细胞(PGCs)来源的小鼠EGCs,通过碱性磷酸酶(AKP)染色、免疫荧光细胞化学、体内分化及体外分化等方法检测EGCs的多能性,并以小鼠胚胎干细胞(ESCs)为对照,应用Real-time PCR检测EGCs中与发育相关的Ins2、Lgf2、H19、Lgf2r等11个父源与母源印记基因的表达情况。结果成功建立小鼠EG细胞系,EGCs克隆AKP染色显示有高水平的AKP活性,免疫荧光细胞化学方法显示克隆表达小鼠ESCs多能性标记物Oct4及细胞表面标记SSEA-1。核型分析检测显示,小鼠EGCs为正常的40条染色体,体内可分化出3个胚层来源的组织,说明小鼠EGCs具有多能性;Real-time PCR结果显示EGCs的印记基因表达量显著高于ESCs。结论12.5dpc PGC来源的EGCs的印记基因处于擦除状态。
Objective To establish mouse embryonic germ cell (EGCs) lines and to analyze the imprinting status of mouse embryonic germ cells. Methods EGCs derived from primordial germ cells (PGCs) at 12.5d (12.5dpc) post-mating were used to detect EGCGCs via alkaline phosphatase (AKP) staining, immunofluorescence cytochemistry, in vivo differentiation and in vitro differentiation Real-time PCR was used to detect the expression of 11 parental and maternal imprinting genes related to development, such as Ins2, Lgf2, H19, and Lgf2r, in mouse embryonic stem cells (ESCs). Results The mouse EG cell line was successfully established. AKP staining of EGCs clone showed high level of AKP activity. Immunofluorescence cytochemistry showed that mouse ESCs pluripotent marker Oct4 and cell surface marker SSEA-1 were cloned and expressed. Karyotype analysis showed that mouse EGCs were normal 40 chromosomes and could differentiate into 3 germ layers in vivo, indicating that mouse EGCs were pluripotent. Real-time PCR results showed that the expression of imprinted genes of EGCs was significantly high In ESCs. Conclusion The imprinted genes of 12.5dpc PGC-derived EGCs are in an erased state.