体外定向诱导胚胎干细胞分化为内皮细胞及其与脐静脉内皮细胞的比较

发布时间：2019-01-24 08:33

【摘要】：目的:研究干细胞定向分化为内皮细胞的效率,并与人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)比较。方法:用两步法诱导干细胞HUES9分化为内皮细胞,前3 d在N2B27培养基中加入骨形态发生蛋白4(25 ng/m L)和肝糖原合成激酶3β受体的选择性抑制剂CHIR99021(10μmol/L)使细胞分化到中胚层状态,第4天开始用VEGF165(200 ng/m L)和Forskolin(2μmol/L)将细胞诱导为内皮细胞。观察分化第6天细胞形态,并与HUVECs比较。用CD144作为内皮细胞表面标志物,流式细胞术检测分化效率及分化后细胞的身份。比较两种内皮细胞迁移和成管能力。结果:分化后的内皮细胞与HUVECs在显微镜下形态一致。分化细胞表面标志物阳性率达到73.4%,重新培养后的内皮细胞表面CD144阳性率达到86.6%,HUVECs为94.4%。分化后的内皮细胞与HUVECs均具有良好的迁移和成管能力,但分化后的内皮细胞能力不如HUVECs强。运用SB431542后,能够提高分化后内皮细胞的迁移和成管能力。结论:此两步法将干细胞分化为内皮细胞有较高的分化效率,可作为理想的分化方法。
[Abstract]:Aim: to study the efficiency of stem cell differentiation into endothelial cells and compare it with human umbilical vein endothelial cell (human umbilical vein endothelial cells,HUVECs). Methods: stem cell HUES9 was induced to differentiate into endothelial cells by two-step method. In the first 3 days, bone morphogenetic protein 4 (25 ng/m L) and CHIR99021 (10 渭 mol/L), a selective inhibitor of hepatic glycogen synthesis kinase 3 尾 receptor, were added to N2B27 medium to differentiate the cells into mesoderm state. On the 4th day, VEGF165 (200 ng/m L) and Forskolin (2 渭 mol/L) were used to induce endothelial cells. Cell morphology was observed on the 6th day of differentiation and compared with HUVECs. CD144 was used as the surface marker of endothelial cells. Flow cytometry was used to detect the differentiation efficiency and the identity of differentiated cells. The migration and tubular ability of two kinds of endothelial cells were compared. Results: the morphology of differentiated endothelial cells was identical to that of HUVECs under microscope. The positive rate of CD144 on the surface of differentiated cells was 73.4%, and the positive rate of CD144 on the surface of cultured endothelial cells was 86.6%. The positive rate of HUVECs was 94.4%. The differentiated endothelial cells and HUVECs had good migration and tube-forming ability, but the differentiated endothelial cells were not as strong as HUVECs. Application of SB431542 can improve the migration and tubular formation of differentiated endothelial cells. Conclusion: the two-step method has higher differentiation efficiency and can be used as an ideal method to differentiate stem cells into endothelial cells.
【作者单位】：中南大学湘雅医院心内科;湖南省人民医院老年病科;
【基金】：国家自然科学基金(81570334)~~
【分类号】：R54

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