人胚胎干细胞的培养条件与鉴别的方法学研究

目的：研究人胚肺成纤维细胞(human Embryonic Lung Fibroblast，HELF)饲养层对人胚胎干细胞(Human Embryonic Stem Cells，hES细胞)生长的作用，摸索适宜的hES细胞培养与识别方法。方法：以流产胎儿肺组织制备HELF饲养层，取生殖嵴或背肠系膜及其周围组织，获得由人原始生殖细胞(human Primordial Germ Cells，hPGCs)转化成的hES细胞。比较其在不同条件下的增殖行为，并通过生物学特性鉴别该细胞系。结果：HELF饲养层在LIF存在的条件下有利于支持hES细胞的体外生长；hES细胞碱性磷酸酶组织化学染色及端粒酶ELISA检测阳性；具有正常的核型；在体外发展为类胚体。结论：HELF饲养层和LIF的协同作用能支持hES细胞的生长；建立通过生物学特性鉴别hES细胞的方法。

Research on the method of culture and identification of embryonic stem cells from human primordial germ cells

Objective:To study the cooperated effect of Human Embryonic Lung Fibroblast(HELF) feeder layer and LIF in the culture of human Embryonic Stem(hES) cells and establish the method of identifying hES cells from Primordial Germ Cells(PGCs).Methods:Embryonic lungs were mechanically disaggregated,then cultured to establish HELF feeder layer.Gonadal ridges and mesenteries of embryos were mechanically disaggeregated,then cultured and passaged in vitro.Comparing the growth characters of hES cells in different conditions.To identify hES cells through their biological characteristics.Results:The coactions of HELF feeder layer and LIF play an important role in proliferation and undifferentiation of hES cells in vitro.High levels of AKP and telomerase activity are associated with hES cells. The cultrued cells have been continuously passaged for more than two months and found to be karyotypically normal and stable.When differentiating,they form embryoid bodies(EBs).Conclusion:To avoid indection of the heterogeneous protein,HELF feeder layer, in the presence of LIF,can be used in culture of hES cells;hES cells from PGCs can be identified through their biological characteristic.