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目的采用简单贴壁培养方法诱导胚胎干细胞(embryonic stem cell,ESC)分化为内皮样细胞,为组织工程血管及细胞替代治疗提供新的种子细胞。方法小鼠ESC株SV129按2×104个/cm2密度传代培养,采用ESC培养基添加1000U/mL白血病抑制因子(leukaemia inhibitory factor,LIF)维持其未分化状态。撤除LIF,ESC悬浮培养形成拟胚体(embryoid body,EB),将培养第4天的EB置于含VEGF的培养基中贴壁培养,诱导分化。采用免疫组织化学染色、流式细胞仪分析、DiI标记的乙酰化低密度脂蛋白(1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeledacetylated low density lipoprotein,DiI-Ac-LDL)摄取实验以及透射电镜检查鉴定分化细胞的性质。结果分化产生的内皮样细胞具有内皮细胞形态特征,能摄取DiI-Ac-LDL。ESC诱导分化培养产生的第15代细胞免疫组织化学染色呈Flk-1和CD31阳性;流式细胞仪检测ESC传至9代后CD31阳性细胞>90%;透射电镜可见怀布尔-帕拉德体及内皮细胞间紧密连接。结论采用简单贴壁培养的方法,单独使用VEGF即可诱导ESC分化为内皮样细胞。诱导培养方法操作简便,经济实用,可为组织工程血管及细胞替代治疗提供所需内皮样细胞。
OBJECTIVE: To induce embryonic stem cells (ESC) to differentiate into endothelium-like cells by simple adherent culture and to provide new seed cells for tissue engineering of vascular and cell replacement therapy. Methods Mouse ESC strain SV129 was subcultured at a density of 2 × 104 cells / cm2, and 1000 U / mL leukemia inhibitory factor (LIF) was added into ESC medium to maintain its undifferentiated state. The LIF was removed and the embryoid bodies were cultured in suspension to form embryoid body (EB). On day 4 of culture, the EBs were cultured in adherent medium containing VEGF and induced to differentiate. Immunohistochemical staining, flow cytometry analysis, DiI-labeled low density lipoprotein (Di-Ac-LDL) uptake by Di-labeled acetylated low density lipoprotein Experiments and transmission electron microscopy were used to identify the nature of differentiated cells. Results Endothelial-like cells produced by differentiation had endothelial cell morphological characteristics and could capture DiI-Ac-LDL. The 15th generation cells induced by ESC induced positive staining of Flk-1 and CD31, the positive cells of CD31 were detected by flow cytometry after passage 9, and the positive cells were found to be> 90% by transmission electron microscopy. And endothelial cells tightly connected. Conclusion Using simple adherent culture method, VEGF can induce the differentiation of ESC into endothelial-like cells. Induction culture method is simple, economical and practical, can provide the desired endothelial-like cells for tissue engineering vascular and cell replacement therapy.