人胚胎干细胞在血清和无血清培养体系中的特性比较  

Characteristic comparison of human embryonic stem cells in serum-containing and serum-free medium

作　　者：胡智兴[1,2] 周轶平[3,2] 吴兰鸥[1] 罗敏[3,2] 郑春兰[1] 李玛琳[3,2] 

机构地区：[1]昆明医学院药理学教研室,昆明650031 [2]昆明医学院云南省高校生物医学工程中心,昆明650031 [3]昆明医学院云南省天然药物药理重点实验室,昆明650031

出　　处：《解剖学报》2010年第3期419-424,共6页Acta Anatomica Sinica

基　　金：云南省自然科学基金项目(2005C0013R);云南省应用基础研究项目(20070044R;2007C0044R)

摘　　要：目的探讨血清培养体系和无血清培养体系对人胚胎干细胞(hES cells)生长特性的影响。方法将人胚胎干细胞株BG02接种在丝裂霉素C处理灭活的小鼠胚胎成纤维细胞饲养层上,分别在含血清hES完全培养基或无血清培养基中连续培养25～30代。在不同培养体系下比较人胚胎干细胞形态、集落贴壁率;运用BrdU掺入法检测人胚胎干细胞增殖,用细胞计数法计算细胞倍增时间;采用免疫荧光染色检测人胚胎干细胞特异性分子标志的表达;用流式细胞仪检测人胚胎干细胞Oct-4,Nanog阳性的比例;RT-PCR检测成纤维细胞生长因子(FGFs)家族基因的表达。结果在两种培养体系的BG02细胞都具有人胚胎干细胞的形态特征。在含血清培养体系下,BG02细胞集落贴壁率和表达OCT-4,Nanog的阳性细胞明显高于无血清培养体系(P<0.05)。无血清培养体系中,BG02细胞生长速度明显高于含血清培养体系,细胞倍增时间分别为(33.8±4.3)h、(45.9±5.7)h,(P<0.05)。无血清培养体系的BG02细胞高表达FGF2、FGFR2、FGFR4。结论两种不同培养体系中人胚胎干细胞的体外培养特性存在一定的差异,可能与BG02细胞FGFs家族基因激活有关。Objective To evaluate the biological characteristic of human embryonic stem （hES） cells in serumcontaining and serum-free culture condition. Methods Human embryonic stem cell line BG02 was cultured on mitomyein C treated mouse embryonic fibroblasts（MEF） feeder layers in medium supplied with serum or knockout serum replacement for 25-30 passages to compare their morphology. The proliferation of hES cells clones was detected by BrdU incorporation assay. The double time of hES cells was evaluated by cell counting. The pluripotency markers of hES cells were examined by immunofluorescence staining. The proportions of Oct-4 and Nanog positive cells were assessed by flow cytometry. Expressions of fibroblast growth factor（FGFs） family genes were determined by RT-PCR. Results Although BG02 cells in the serum and serum-free culture medium shared similar morphology, hES clones in serum-free culture condition showed more typical morphology and more Oct-4, Nanog positive cells than that in serum-containing condition. The attachment rate of hES cells clumps cultured in serum-free condition was obviously higher than that of serum condition （P 〈 0.05）. The growth of BG02 cells in serum-containing medium was more fast （P 〈 0. 05）. The doubling time of hES cells in the serum and serum-free condition was （45.9 ± 5.7） hours and （33.8 ± 4. 3 ） hours, respectively. Additionally, BG02 cells in serum-free culture condition expressed high level of FGF2, FGFR2, and FGFR4. Conclusion BG02 cells cultured in serum or serum-free culture condition represent some variations in the growth and characteristics of hES cells. These variations might be attributed to the activation of members of FGFs family in BG02 ceils.

关 键 词：胚胎干细胞 血清 BrdU掺入法 免疫荧光 人 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]