人脐静脉内皮细胞饲养层促进胚胎干细胞的生长

Human umbilical vein endothelial cells as a feeder layer promote the growth of embryonic stem cells

目的探讨人脐静脉内皮细胞是否可作为饲养层支持胚胎干细胞（ESCs）的生长。方法分离培养人脐带内皮细胞，采用生长良好的3代内皮细胞，灭活后制备饲养层，通过观察碱性磷酸酶染色、胚胎干细胞特异性表面标志物检测、染色体核型分析和严重联合免疫缺陷小鼠（SCID）体内畸胎瘤形成实验，对在内皮细胞上的E14．1胚胎干细胞进行鉴定。结果E14．1胚胎干细胞在人脐静脉内皮细胞上传3～8代后细胞呈克隆性生长，高度表达碱性磷酸酶、SSEA-1及Oct-4。传15代后仍表现正常的二倍体核型。传20代的ESCs接种到SCID小鼠，6周后均能形成畸胎瘤。结论人脐静脉血管内皮细胞能有效地支持ESCs的生长，解决了ESCs临床应用的生物安全性问题。

Objective To investigate whether human umbilical vein endothelial cells as a feeder layer was capable of supporting the growth of embryonic stem cells in vitro. Methods Human umbilical vein endothelial cells were isolated and cultured and then prepared as feeder cells after 3 passages. Alkaline posphatase activity （AKP） staining, stem cell surface marker test and karyotypes were conducted in different periods of cell culture. The suspension of stem cells cultured after 20 passages on endothelial cells were inoculated to the legs of severe combined immunodeficiency （SCID） mice subcutabeously to test the teratoma formation. Results E14. 1 embryonic stem ceils retained good colonies when they were cultured on endothelial cells for 3 passages and 8 passages. In addition, they expressed SSEA-1, Oct-4, and a membrane alkaline phosphatase to a high extent at passages 3 and 8. Embryonic stem cells cultured for 15 passages stably retaind a normal karyotype. Embryonic stem cells cultured on endothelial cells for 20 passages were inoculated into the hind leg of SCID mouse, teratomas containing three embryonic layers were recovered six weeks later. Conclusion Human umbilical vein endothelial ceils would support effectively embryonic stem cells expansion, and provide a clinically safe method to expand ES cells for future clinical application.