摘要
目的建立人胚胎干细胞无动物源性饲养层培养方法,同时对长时间体外培养的人胚胎干细胞核型变化进行分析。方法人胚胎干细胞系HUES4细胞分别培养于小鼠胚胎成纤维细胞和人包皮成纤维细胞饲养层,并对其干细胞特性进行鉴定;在培养传代过程中,收获P27、P34、P41和P44细胞进行染色体核型分析,P27细胞还进行DNA短串联重复序列多态性分析。结果生长于人包皮成纤维细胞饲养层的HUES4细胞碱性磷酸酶染色以及SSEA-4、TRA-1-60和TRA-1-81抗原阳性,SSEA-1抗原阴性。所检测的4代细胞中均见46,XY/46,XY,t（9;15）（q22;q26）核型嵌合现象,且异常核型百分比随传代次数增加有上升的趋势。结论培养人胚胎干细胞的饲养层细胞可由无动物源性的饲养层细胞替代;长期体外培养有增加细胞染色体核型异常的风险。
Objective To establish a protocol for culturing of human embryonic stem cells （HUES4） without any animal-derived feeder cells and to investigate the karyotype stabilities of HUKS4 cells after long-term cultivation. Methods HUKS4 cells were cultured on mitomycin C treated MEFs or human foreskin flbroblast feeder cells. The pluripotency of the KS cells was analyzed by immunocytochemistry staining to detect the expression of pluripotent marker, karyotype of the KS cells at passage 27, 34, 41, 44 and short tandem repeat （SIR） at passage 27 were analyzed： Resalts The HUES4 cells cultured on human feeder cells were positive for alkaline phosphatase activity, SSEA-4, TRA-1- 60 and TRA-1-81 staining, but negative for SSEA-1. Analysis of karyotype at different passages suggested an abnormal karyotype 46, XY, t（9; 15 ） （ q22; q26） mosaicism occurred in HUES4, and the ratios of abnormal increased with passage. Conclusion HUES4 could be cultured without animal-derived feeder cells and the incidence of abnormal karyotype might be increased with long-term culture.
出处
《中华医学遗传学杂志》
CAS
CSCD
北大核心
2007年第3期275-278,共4页
Chinese Journal of Medical Genetics
基金
广州市科技局重大科技攻关项目（200621-E0021）
