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背景:到目前为止,国际上通用的动物胚胎干细胞细胞系大多是从129品系和C57BL/6系小鼠中获得的,少部分来自BALB/C系,来自于中国昆明鼠则鲜有报道。目的:分离培养昆明系小鼠胚胎干细胞,并对其生物学特性进行初步鉴定。设计、时间及地点:以细胞为对象的观察性实验,于2006-05/2007-06在南昌大学第一附属医院泌尿外科研究所完成。材料:昆明小鼠3.5d胚龄的囊胚。方法:自昆明系小鼠3.5d胚龄的囊胚分离内细胞团细胞,接种于小鼠原代胚胎成纤维细胞饲养层上,对分离的内细胞团细胞进行扩增、传代培养。主要观察指标:观察胚胎干细胞集落的生长情况,对其碱性磷酸酶表达进行染色分析,采用免疫荧光法检测其阶段特异性胚胎抗原1表达,以反转录-聚合酶链反应检测其特异性表达因子c-Myc,Nanog,Oct3/4和Sox2的基因表达情况,并对其体内外分化能力对进行鉴定分析。结果:分离、培养自内细胞团的细胞呈典型的胚胎干细胞集落样生长,碱性磷酸酶染色及阶段特异性胚胎抗原1荧光染色阳性,细胞表达c-Myc,Nanog,Oct3/4和Sox2等基因,细胞在体内外均能分化成来源于不同胚层的多种细胞类型。结论:成功从昆明小鼠囊胚中分离并建立一株胚胎干细胞系,其生物学特性与其他胚胎干细胞株相符。
BACKGROUND: So far, most of the commonly used animal embryonic stem cell lines in the world are obtained from 129 and C57BL / 6 mice, and a few are derived from BALB / c lines. However, Kunming mice from China are seldom reported. Objective: To isolate and culture Kunming mouse embryonic stem cells and identify its biological characteristics. DESIGN, TIME AND SETTING: Observational experiments targeting cells were performed at the Institute of Urology, the First Affiliated Hospital of Nanchang University from May 2006 to June 2007. MATERIALS: Kunming mice blastocysts aged 3.5 days. Methods: The inner cell mass cells were isolated from the blastula of Kunming mice aged 3.5 days and inoculated on the feeder layer of mouse primary embryonic fibroblasts. The isolated inner cell mass cells were amplified and subcultured. MAIN OUTCOME MEASURES: The growth of embryonic stem cell colonies was observed. The alkaline phosphatase expression was analyzed by immunofluorescence. The expression of stage specific embryonic antigen 1 was detected by immunofluorescence. The specificity of reverse transcription - polymerase chain reaction Expression of c-Myc, Nanog, Oct3 / 4 and Sox2 gene expression, and its ability to differentiate in vitro and in vivo were identified and analyzed. Results: The cells isolated and cultured from inner cell mass were typical of ESCs colony - like growth. Alkaline phosphatase staining and stage - specific embryonic antigen 1 staining were positive. The cells expressed c - Myc, Nanog, Oct3 / 4 and Sox2 Genes and cells can differentiate into many cell types derived from different germ layers both in vitro and in vivo. CONCLUSION: An ES cell line was successfully isolated from Kunming mouse blastocysts and its biological characteristics were in accordance with those of other embryonic stem cell lines.