人胚胎生殖系细胞分离与体外培养的初步研究  

Isolation and cultivation of human embryonic germ cells

作　　者：刘善荣[1] 刘厚奇[1] 汤淑萍[1] 惠宁[2] 冀凯宏[1] 熊俊[1] 蒋正[2] 戚中田[3] 

机构地区：[1]第二军医大学基础医学部组织胚胎学教研室,上海200433 [2]第二军医大学长海医院妇产科 [3]第二军医大学基础医学部微生物学教研室

出　　处：《第二军医大学学报》2003年第9期941-943,共3页Academic Journal of Second Military Medical University

基　　金：国家杰出青年科学基金 ( 3 982 5 116) ;海外青年学者合作研究基金 ( 3 992 80 0 9)

摘　　要：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 .2 5 %胰酶消化 3min ,种植到丝裂霉素C处理过的小鼠胚胎成纤维细胞上培养 3d ,挑取克隆传代 ,在第 1 0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 。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.

关 键 词：胚胎 生殖系细胞 细胞分离 体外培养 碱性磷酸酶 

分 类 号：R329.21[医药卫生—人体解剖和组织胚胎学] R321.1[医药卫生—基础医学]