胚胎干细胞分化形成囊状胚胎小体的形态学变化特征

目的观察胚胎干细胞R1(ESs)体外分化形成囊状胚胎小体(CEBs)及其血管内皮细胞和平滑肌细胞的时相规律和结构特点,探讨其在血管发育生物学研究中的作用和意义。方法体外悬浮培养ESs形成CEBs,采用HE染色观察CEBs的结构特点。用免疫荧光染色鉴定不同培养时间的CEBs中CASPASE-3的表达。加入全反式维甲酸(atRA)及联丁酰基cAMP(DBcAMP)定向诱导ESs分化,其后行平滑肌特异性标志物α-肌动蛋白(SMα-actin)及内皮细胞特异性标志物PECAM-1免疫荧光染色。结果典型的CEBs在第13d左右可以形成。第6d就可见CEBs中心部位出现调亡细胞,坏死腔开始形成,到第13d时中心坏死腔完全形成,凋亡细胞明显增多。将第6d的CEBs接种于培养皿后用atRA及DBcAMP处理,3d后可见SMα-actin染色阳性细胞。而未经处理的CEBs贴壁后,在第10d可见PECAM-1的表达。结论CEBs能够模拟体内血管发生形成的过程,可作为研究血管发育生物学的较好模型。

CHARACTERS OF MORPHOLOGICAL CHANGE IN CYSTIC EMBRYOID BODIES DIFFERENTIATED FROM EMBRYONIC STEM CELLS

Objective To examine the phase regularity and structural characters of cystic embryoid bodies (CEBs), endothelial cells and smooth muscle cells differentiated from mouse embryonic stem cells R1 (ESs) in vitro, in order to elucidate the function and significance of this model in the study of vascular developmental biology.Methods CEBs were formed from mouse ESs cultured in suspension in vitro. HE staining was performed to observe structural characters of CEBs at different time points.Caspase-3 immunofluorescence was examined by confocal microscopy in CEBs at different time points.ESs were induced to differentiate specifically by the addition of all-trans retinoic acid (atRA) and dibutyryl-cyclic adenosine monophosphate(DBcAMP). The expressions of smooth muscle specific alpha-smooth muscle actin and endothelial specific PECAM-1 were examined by immunofluorescent staining.Results Typical CEBs were formed on day 13. On day 6 some central cells of CEBs underwent apoptosis, and necrotic cavities appeared. A single central necrotic cavity emerged on day 13, and apoptosis was more conspicuous. The CEBs on day 6 were treated with atRA and DBcAMP, and the expression of smooth muscle specific alpha-actin was detected after 3 days.PECAM-1 immunoreaction was present on day 10 in plated CEBs without atRA and DBcAMP treatment. Conclusion CEBs can simuate early in vivo vasculogenesis, accordingly this in vitro model may be used for the research of vascular developmental biology.