Isolation,cultivation and identification of human embryonic fibroblasts and expressions of cytokines essential for growth of human embryonic germ cells in vitro / 吉林大学学报(医学版)

ABSTRACT

Objective To isolate,cultivate and identify the human embryonic fibroblasts(hEFs) derived from the gonadal ridges and dorsal mesenteries of human embryos,and to detect the expression by hEFs of cytokines crucial for the growth of human embryonic germ cells(hEG) in vitro.Methods The hEFs were isolated by enzyme digestion from gonadal ridges and dorsal mesenteries of 5-to 9-week old human embryos.The cells were then cultivated.The biologic characteristics(morphology,growth characteristics and cell cycle) of these cells were also studied.The reverse transcription-polymerase chain reaction(RT-PCR) was used to seek the expressions of a specific fibroblast marker,prolyl 4-hydroxylase ?,and a specific marker of epithelial cells,cytokeratin-4,in the cells.The expressions of cytokines essential for the growth of hEG,namely basic fibroblast growth factor(bFGF) and leukemia inhibitory factor(LIF),were also examined by RT-PCR.Results The hEFs were successfully isolated and cultivated from gonadal ridges and dorsal mesenteries of human embryos.They could be passaged beyond the 25th generation.The biologic characteristics of the cells did not change,even in high-passage cells or frozen-thawed cells.The cells expressed prolyl 4-hydroxylase ?, but not cytokeratin-4,which was similar to the fibroblasts.The cultured cells expressed bFGF and LIF.Conclusion The hEFs derived from gonadal ridges and dorsal mesenteries of human embryos are successfully isolated and cultivated,and the cells express the cytokines essential for the growth of hEG in vitro.