摘要
[目的]探讨分离培养人原始生殖细胞的最佳条件。[方法]用胰蛋白酶消化5～9周人流组织中的人胚生殖嵴,获取人原始生殖细胞。以小鼠胚胎成纤维细胞作饲养层,在高糖DMEM培养基中添加10μmol/L福司克林(forskolin)和5～μg/L碱性成纤维细胞生长因子(bFGF)的培养液中培养、传代,并对子代细胞进行碱性磷酸酶活性检测和体外分化实验。[结果]原代培养时形成许多大小不等,形态各异的由人原始生殖细胞组成的集落,约5～7d传代。传代后,集落生长,变大。细胞培养到第七代,检测碱性磷酸酶染色为强阳性,体外分化实验有拟胚体形成。[结论]人原始生殖细胞可以用胰蛋白酶消化分离培养。用高糖DMEM培养基,添加forskolin和bFGF有利于人原始生殖细胞增殖。体外分化实验初步证实人原始生殖细胞具有多向分化潜能。
[Objective]To establish a proper system for the isolation and ideal culture conditions for human primordial germ cells (hPGCs ) in vitro. [ Methods]hPGCs were isolated from the gonadal ridges of 5 - 9 weeks old abortion postfertilization human embryonic tissues and digested with trypsin, then cultured in high glucose DMEM (H-DMEM) containing 10μmol/L forskolin and 5 - 10μg/L human recombinant basic fibroblast growth factor (bFGF). Mouse embryonic fibroblast was used as feeder layer. The morphology was observed under light microscopy. The alkaline phosphates (AKP) test and differentiation test in vitro were carried out. [Results] Colonies of hPGCs cells with different sizes and morphology were well formed in primary culture. In passage cultured, colonies grew well and became somewhat bigger than that in primary culture and could be subcultured one generation in 5 - 7 d. Until 7th generation, hPGCs expressed AKP strong positive staining and gave rise to embryoid bodies in vitro. [Conclusion]The results indicate that hPGCs can be isolated and cultured with trypsin. The H-DMEM medium containing forskolin and bFGF can maintain survival and proliferation of hPGCs effectively. Primary experiment in vitro shows that the hPGCs have multiple differentiation potential.
出处
《中山大学学报（医学科学版）》
CAS
CSCD
北大核心
2003年第4期325-328,共4页
Journal of Sun Yat-Sen University:Medical Sciences
基金
国家重点基础研究课题(973)基金(G1999054301-2)
