体外定向诱导小鼠胚胎干细胞向内皮细胞分化的研究

目的探讨体外定向诱导胚胎干细胞(embryonic stem cell,ESC)向内皮细胞分化的条件,为组织工程血管提供种子细胞。方法取怀孕12.5d的昆明小白鼠1只,断颈处死,取其胚胎,培养制备小鼠胚胎成纤维细胞(mouse embryonic fibroblast,MEF)。常规复苏ESC后进行体外培养,采用悬滴-悬浮法制备拟胚体(embryoid body,EB)。将EB分两组:实验组加入含有3ng/ml转化生长因子β1、50ng/ml血管内皮细胞生长因子及1μmol/L激活素受体样激酶选择性抑制剂的EB培养基;对照组只加入EB培养基。倒置显微镜下观察细胞生长情况。采用RT-PCR及免疫组织化学染色检测诱导细胞vWF和CD34的表达,验证细胞性质。结果原代MEF生长迅速,第3天细胞融合达90%左右,细胞呈长梭形,有少量侧支,细胞核饱满,有2~3个核仁,细胞排列紧密。传至3~5代,细胞呈多角形,胞浆饱满,有3~4个核仁,细胞表面分泌较多细胞颗粒。ESC在饲养层上保持未分化状态,细胞团形态呈鸟巢样,边缘平整,单个细胞体积小,折光性强,核质比高,增长速度快。悬滴培养3d的EB肉眼可见,再经悬浮培养3d后,形成较大的透亮EB;EB贴壁后第2天,球体略摊开。实验组第4~7天,EB周围有许多圆形细胞产生,第10~14天,可见由大量圆形细胞组成的血管样结构自EB周围生长;对照组EB周围未见血管样结构生长。免疫组织化学染色见EB周围大量vWF染色阳性细胞,RT-PCR检测到vWF和CD34的表达。结论ESC在特定的条件下可以分化为内皮细胞,有可能为组织工程血管提供大量的种子细胞。

INDUCED DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELL INTO ENDOTHELIAL CELL IN VITRO

OBJECTIVE: To explore an optional condition to induce mouse embryonic stem cell (ESC) to differentiate into endothelial cells so as to provide seed cells for tissue engineered vascular. METHODS: The embryos from one pregnant 12.5-days mouse was harvested to culture the mouse embryonic fibroblasts (MEF). The ESC was reanimated by common method, and used to cultured into embryoid body (EB) in vitro. The EB which was used to induce into endothelial cells was divided into two groups. The EB was cultured in the EB medium with 3 ng/ml transforming growth factor beta1, 50 ng/ml vascular endothelial cell growth factor and 1 micromol/L potent and selective inhibitor of activin receptor-like kinase receptors in experimental group. The EB was cultured in the EB medium in the control group. After 14 days, RT-PCR and immunohistochemistry were used to detect vWF and CD34, to analyze the morphology and type of the differentiated cells from ESC. RESULTS: The primary MEF had a high proliferation activity. At the 3rd day, the fusion rate of MEF was about 90% with a fusiform shape. The cells was fusiform shape and arranged compactly with fullness of nucleus and 2-3 entoblasts. The 3rd-5th generations EB was polygonal with fullness of cytoplasm and 3-4 entoblasts. ESC could maintain undifferentiated state, and the cells unit looked like bird nest with smooth margin; the cells was small at size and strong refractivity with high rate of nuclein and rapid proliferation. At 3 days of drop-culture, EB can seen grossly and at 3 days of suspension, large and transparent EB formed. EB was spread radiately with an intensive adhesion at the 2nd day. In experimental group, many round cells was differentiated around EB from the 4th day to the 7th day, and form tubular structures from the 10th day to the 14th day. The vWF and CD34 were expressed. In control group, EB could not form tubular structures, and the vWF and CD34 were not expressed. CONCLUSION: ESC can differentiate into endothelial cells under some conditions, and form vessel-like structure under condition culture, which can provide sources of seed cells for tissue engineered vessel.