利用小鼠胚胎干细胞贴壁制备拟胚体的新方法研究

A novel method for preparing embryoid bodies with attached embryonic stem cells

目的为改进拟胚体制备过程中分化不同步等问题,探讨建立小鼠胚胎干细胞贴壁制备拟胚体的新方法。方法沈阳军区总医院心内科于2004年10月至2006年1月对小鼠进行研究,当小鼠胚胎干细胞R1生长至70%～80%亚融合时,将其消化成单细胞,以1×106的细胞数接种到直径100mm组织培养皿中,用含低浓度白血病抑制因子(LIF)1μg/L的胚胎干细胞培养液连续贴壁培养3d后,收集贴壁细胞团继续悬浮培养。对悬浮培养的拟胚体及其冰冻切片行形态学观察,对悬浮及贴壁分化的拟胚体行甲胎蛋白(AFP)、血小板内皮细胞粘附分子-1(PECAM-1)及神经丝蛋白68KD(NF-68)免疫荧光染色。结果形态学观察用本法得到的拟胚体大小均一,分化同步,能展示从简单拟胚体到成熟囊性拟胚体的典型发育过程。免疫荧光染色显示,AFP、PECAM-1及NF-68三种标志物表达均为阳性。结论与传统方法相比,该法操作简便、拟胚体形成率高、分化阶段同步,具有分化为3个胚层来源细胞的能力,可作为胚胎早期发育和胚胎干细胞分化等研究的理想工具。

Objective To improve disadvantages such as asynchrony during embryoid bodies （EBs） preparation, and to establish a novel method to prepare EBs by using attached mouse embryonic stem Cells. Methods Mouse R1 embryonic stem （ES） cells were trypsinized to a single cell suspension when reaching a sub -confluent state of 70 % -80 %, then 1 ×10^6 ES cells were plated into 100 mm tissue culture dishes. After being cultured in medium containing low concentration leukemia inhibitory factor （LIF, 1 μg/L） for 3 days, EBs were collected and suspended in EBs culture medium for further development. Morphology studies were performed on suspended EBs and their cryosections, then immunofluorescent staining of α - fetoprotein （ AFP）, platelet endothelial cell adhesion molecule - 1 （ PECAM - 1 ）, and neurofilament 68 KD （ NF - 68） were performed on suspended and attached EBs. Results EBs obtained by this method were homogeneous in size, synchronous in developmental stage; typical in structure and could well display the developmental process from simple EBs to mature cystic EBs. Immunofluorescent staining showed that AFP, PECAM - 1 and NF -68 were positive. Conclusion Compared with present methods of preparing EBs, this novel method have advantages of simple manipulation, high efficiency of EBs formation and obtaining EBs at synchronous developmental stage, furthermore, these EBs have typical structures and the potential to differentiate into derivatives of all three embryonic germ layers. Thus this method can be used as an ideal tool for studies on early embryonal development, ES cell differentiation and so on.