人胚胎生殖系细胞分离与体外培养的初步研究

目的:初步探讨人胚胎生殖系细胞的分离与体外培养.方法:体外分离人胚胎生殖嵴和肠系膜组织,按单纯机械分离、酶消化分离、二者结合法及是否有饲养层将组织分为5种不同的方式培养后,用碱性磷酸酶标记,计数阳性克隆并进行比较.结果:酶消化法培养所获得的克隆较单纯机械分离方法多,原代培养时种植到饲养层上效果较好.将分离到的组织用0.25％胰酶消化3 min,种植到丝裂霉素C处理过的小鼠胚胎成纤维细胞上培养3 d,挑取克隆传代,在第10天形成30.7 个阳性克隆,显著高于其他方法获得的阳性克隆数.用碱性磷酸酶标记的细胞(克隆)具有多样性,细胞胞体为圆形,分为有突起和无突起两种;阳性细胞克隆呈圆球形、条带形和块形,与周围细胞分界清楚.结论:人胚胎体内的微环境限制生殖系细胞的体外扩增培养,原代培养需要在饲养层上进行且宜用酶消化法尽早传代.

Isolation and cultivation of human embryonic germ cells

Objective:To study the isolation and in vitro cultivation of human embryonic germ cells. Methods:Human embryonic tissues containing genital ridges and dorsal mesenteries were isolated and then cultured in 5 different patterns: simple mechanical disaggregation,trysinization, combination of the former 2 and with/without feeder cells. Alkaline phosphatase (AP) activity was used as detection marker and positive clones were counted. Feasibility of different patterns was estimated. Results: More clones were found when treated with trypsinization than with the other 2 methods,and it was better when transplanted on feeder cells in primary culture. The 4th method was the best,in which the tissues were incubated with 0.25% trypsin for 3 min and then cultured on a mouse fibroblast feeder layer mitotically inactivated for 3 d.Clones were picked out and passaged and 30.7 clones were found after cultured for 10 d. The cells with high level of AP activity were of multiformity; cell body was round with or without dendrites. Positive cell clones were spherical, strapping and patch with a significant ambit. Conclusion:Human embryonic microenvironment in vivo restricts germ cells from proliferating in vitro . Feeder cells should be plated in primary culture and the culture should be passaged by trypsinization as early as possible.