学术
稳定表达GFP的胚胎干细胞株的建立及其生物学特性研究
钱坤[1] 陈红[2] 朱桂金[1] 张苏明[3]
[1]华中科技大学同济医学院附属同济医院生殖医学中心,430030 [2]华中科技大学同济医学院附属同济医院康复医学科,430030 [3]华中科技大学同济医学院附属同济医院神经科,430030
摘 要:
目的建立稳定表达绿色荧光蛋白（GFP）的胚胎干细胞株,为探索胚胎干细胞及其衍生细胞移植后在体内的分化、迁移及整合提供细胞模型。方法构建含Neo抗性基因质粒pCX-EGFP-Neor并用脂质体转染胚胎干细胞系ES-D3,G418筛选得到稳定表达的细胞株。细胞计数检测其倍增时间、流式细胞仪分析细胞周期,酶组织化学检测碱性磷酸酶（AKP）的表达,免疫组化检测阶段特异性胚胎抗原-1（SSEA-1）并观察其体外分化能力。结果该细胞株可在体外形成拟胚体及各种形态的成熟细胞且均可发出绿色荧光。该细胞株的倍增时间约为12h,G0/G、G2/M、S期分别为22.16±2.03%、18.46±1.54%、59.38±5.76%。倍增时间和细胞周期与未转染GFP的ES-D3相比没有显著性差异（P〉0.05）。碱性磷酸酶（AKP）和SSEA-1呈强阳性表达。结论我们成功地建立了稳定表达GFP的胚胎干细胞株,且其生物学特性未受到GFP的影响。[著者文摘]
文章出处:
《中国优生与遗传杂志》-2007年15卷10期 -92-95页
栏目信息:
分 类 号:
文献标识码:
A
文章编号:
1006-9534（2007）10-0092-03
Study on the establishment of a mouse embryonic stem cell line ant its biological character under the stably expresse GFP.
QIAN Kun, et al. （ The Productive Center of Tonal Hospital, Huazhong Science and Technique University, 430030）
Abstract:
Objective： To establish a mouse embryonic stem cell （ESC） line which stably expresse GFP and supply a cell model for research on ESC differentiation, migration and integration in vivo. Methods - Inserted Neo resistant gene into plasmid pCX - EGFP and transfected ESC by Lipofectamine, screened ESCs with G418, detected doubling time by cell counting, cell cycle by flow cytometry, AKP by enzyme tissue chemistry and SSEA - 1 by immunochemistry and observed its differentiation ability in vitro. Results ： The GFP^＋ ESCs could differentiate into green embryo bodies and lasted into green matured cell. The doubling time of this cell line was about 12h and the cell cycle distribution was 22. 16 ±2.03% （GO/G）, 18.46 ±1.54% （G2/M）, 59. 38 ±5.76% （S） respectively. The doubling time and cell cycle had no significant difference compared to GFP- ES - D3. Expression of AKP and SSEA - 1 of the GFP ^＋ cell line were both strongly positive, Conclusion： We successfully established a GFP ^＋ ESCs line and it had similar biology character with GFP- ESC lines.[著者文摘]
Key words:
Embryonic stem cell; Green fluorescence protein; Biology character
基金资助:
国家自然科学基金（编号：30600188）
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