经Flk1前祖细胞胚胎干细胞分化为内皮细胞的研究

Differentiation of mouse embryonic stem cells into endothelial cells through Flkl-expressing mesoderm progenitor cells

目的掌握小鼠胚胎干细胞（ESCs）向内皮细胞分化方法，为临床心血管疾病治疗研究的发展奠定基础。方法分化前检测ESCs自我更新标记分子的表达。单层分化4d检测血管内皮生长因子受体2（Flk1）的表达。分化8d检测血管内皮钙黏蛋白（VE—cadherin）的表达，同时于显微镜和透射电镜下观察分化细胞形态。最后在血管内皮生长因子（VEGF）诱导组分化2、4、6、8、10d检测八聚体结合转录因子4（Oct4）、Flk1和vE—cadherin的表达。结果分化前自我更新标记分子的表达结果说明E14细胞具有自我更新能力。分化4d表达Flk1，说明E14细胞分化为中胚层前祖细胞；分化8dVE—cadherin的表达说明E14细胞分化成为血管内皮细胞。分化8d光镜下观察内皮细胞特征性呈“鹅卵石”样结构非常明显；透射电镜下可观察到韦-帕小体（W—P）小体，以上结果说明E14细胞分化成为内皮细胞。最后，对分化用VEGF诱导组分化2、4、6、8、10d的Oct4、Flkl和VE—cadherin表达范围有了掌握。结论单层分化8d，VE—cadherin的表达说明E14细胞已分化为成熟血管内皮细胞。掌握ESCs向血管内皮细胞定向分化方法为临床心血管疾病治疗研究的发展奠守基础。

Objective To master the technique of mouse embryonic stem （ES） cells differentiate into endot- helial cells, which would be a new therapeutic approach for cardiovascular disease. Methods Expression of self- renewal marker genes in E 14 cells was assessed. Expression of vascular endothelial growth factor receptor 2 （Flk 1） in monolayer differentiation on day 4 and vascular endothelial cadherin （VE-cadherin） on day 8 were detected. On day 8, differentiation cells were also observed under phase contrast microscopy （PCM） and transmission electron microscope （TEM）. ES cells and endothelial-specific molecular markers were assessed by RT-PCR at different time-polnts. Results As self-renewal marker genes were expressed in El4 cells, El4 cells was identified to maintain their self- renewal pluripoteney. The marker gene of letarl, Flk 1 was expressed on differentiation day 4. On differentiation day 8 the marker gene VE-cadherin was expressed and as observed under PCM endothelial cells with spindle shape and TEM with Weibel-Palade body, thus were the major populations generated after VEGF induction, and El4 cells were confirmed differentiated into mature endothelial cells. The expressions of genes octamer binding transcription factor 4 （Oct4）, Flkl and VE-cadherin were detected on differentiation day 2, 4, 6, 8 and 10. Conclusions As VE- cadherin gene was expressed in monolayer on differentiation day 8, E14 cells were confirmed differentiated into endothelial cells, which would be a new therapeutic approach for cardiovascular disease.