建立仿人类胚胎早期血管发生的芽生类胚体模型

目的 以分化后人胚胎干细胞为材料,建立仿人类早期胚胎血管发生的芽生类胚体模型.方法 将未分化人胚胎干细胞以类胚体(EB)的形式诱导分化,再以不同的血管生成刺激因子作用于类胚体、进行芽生类胚体的二次诱导,摸索芽生类胚体的最佳培养条件;并以LDL吞噬法、免疫荧光抗体标记以及培养体系中加入血管生成抑制因子等方法检测芽生类胚体中是否存在人胚胎干细胞分化后的早期血管内皮细胞.结果 (1)分化第11天的类胚体已能够在胶原基质中形成芽状分支,彼此间还能互相连接形成网络状结构.(2)在含有血管内皮生长因子(VEGF)、酸性成纤维细胞生成因子(FGF)的胶原基质中,从EB中伸展出的芽状分支能吞噬Dil-AcLDL、表达CD31、具有类似于血管腔的三维中空结构,称为芽生EB.刺激芽生EB发生的细胞因子最佳浓度为:VEGF50ng/ml,FGF 100 ng/nd.(3)芽生EB间的网络状结构,能被血管生成抑制因子内皮抑素和PF4抑制.结论 芽状结构中含有由人胚胎干细胞分化而来的内皮细胞,芽生EB是研究人类胚胎发育过程中早期胚胎血管生成的一种模型.

Establishment of sprouting embryoid body model mimicking early embryonic vasculogenesis in human embryo

Objective To establish a sprouting embryoid body model mimicking early embryonicvasculogenesis in human embryo. Methods Human embryonic stem were (hESCs) were cultured on themouse embryo fibloblasts and then were induced to differentiate to form three-dimensional EB. The hEBswere cultured in media containing various angiogenesis-related factors: vascular endothelial growth factor(VEGF), fibroblast growth factor (FGF), endostatin, angiostatin, and platelet factor (PF) -4 of differentconcentrations for 3 days to observe the sprouting of the hEBs. 3,3,3', 3'-tetramethylindo-carbocyanineperchlorate labeled acetylated low density lipoprotein (Dil-AcLDL) was added onto the hEBs foe 4 hImmunofluorescence assay was used to observe if Dil-AcLDL was absorbed and if CD31 was expressed so asto determine the existence of embryonic endothelial cells in the sprouting structures. The ideal culturingcondition was analyzed. Results The differentiated EBs formed sprouting structures in the collagen Ⅰmatrix containing VEGF and FGF. The sprouts among individual EBs were able to link to each other andform vascular network-like structures. In the presence of VEGF and FGF, the sprouts branching from theEBs assimilated DiI-AcLDL, expressed CD31 and formed a 3-dimensional cylindrical organization. Theconcentrations of growth factors ideally stimulating sprouting growth were 100 ng/ml of VEGF and 50 ng/mlof FGF. The networks among the EBs were abolished by the angiostatin, endostatin, and PF4. ConclusionThe sprouting from hEBs accumulates embryonic endothelial cells and the sprouting network-like structuresare indeed endothelial in nature. Inducing of sprouting EBs is an ideal model that mimics early embryonicvasculogenesis in humans.