两种培养条件下人胚胎干细胞生长状况的比较  

Growth conditions of human embryonic stem cells in two different culture systems

作　　者：蒋伟民[1,2,3] 赵星[1,2] 李崎[1,2] 张宇[1,2] 周知[1,2] 马宁[1,2] 李林江[1,2] 徐雯[1,2] 卢伟英[1,2] 黄元华[1,2] 马燕琳[1,2] 

机构地区：[1]海南医学院附属医院生殖医学中心,海南海口570102 [2]海南省人类生殖与遗传重点实验室,海南海口570102 [3]贵阳中医学院,贵州贵阳550002

出　　处：《海南医学院学报》2013年第9期1175-1179,共5页Journal of Hainan Medical University

基　　金：国家科技部973项目(2012CB966500);海南省科技厅国际合作重点项目(GJXM20100004;GJXM201106);人事部2011年度留学人员人才启动项目;海南省自然科学基金项目(812185);海口市重点科技计划项目(2012078)~~

摘　　要：目的:比较胚胎干细胞株在两种不同培养体系下生长状况,验证两种不同培养体系对于人胚胎干细胞全能性的维持与延续。方法:将人类胚胎干细胞株分别接种于传统的培养体系以及mTESR1的培养体系,观察在不同培养体系下人类胚胎干细胞状态及克隆形态之间的差别。对生长于不同培养体系下的胚胎干细胞进行碱性磷酸酶染色、免疫荧光染色以及用RT-PCR的方法检测人胚胎干细胞全能性。结果:生长在传统培养体系中的胚胎干细胞,细胞克隆薄且扁平,与周围的饲养层细胞边界清晰,克隆中心的细胞容易出现分化。单个胚胎干细胞核质比高,核仁明显。生长mTESR1体系中的胚胎干细胞,细胞克隆较传统培养体系下的大,呈单层细胞生长,细胞排列紧密,单个胚胎干细胞的细胞核大,核仁明显,核质比高。生长在不同培养体系下的人胚胎干细胞碱性磷酸酶染色均呈现阳性,免疫荧光染色及RT-PCR均验证了细胞的全能性。结论:两种不同培养体系均能维持胚胎干细胞的全能性。mTESR1培养条件更加简便,能更清楚观察与分析单个细胞的形态,适合人胚胎干细胞的维持培养。Objective：To compare the growth conditions of human embryonic stem cells in two different culture systems and to verify the capability of these two different culture systems in maintaining the totipotency of human embryonic stem cells.Methods：Human embryonic stem cells were cultured under two different culture systems.Cell growth conditions and the difference in cell colony in two different culture systems were observed.Totipotency of cells growing under two different culture systems were tested through alkaline phosphatase staining assay,immunofluorescent staining assay and RT-PCR assay.Results：In conventional culture systems,the colony was thin and flat and had a clear edge to the feeder cells,and it was easily for the cells in the center of the colony to differentiate.Single stem cell had a high nucleocytoplasmic ratio,the nucleolus was obvious.In mTESR1culture system,the colony was bigger,which grew as monolayer with uniform morphology,stem cells packed closely.Single stem cell had a large nucleus.The nucleolus was obvious and the cells had a highnucleo-cytoplasmic ratio.In verification tests,cells growing under both culture systems were positive in AP staining assay.In immunofluorescent staining assay,cells were positive in the expression of totipotent markers in both culture systems.In RT-PCR assay,cells expressed the totipotent genes in both culture systems.Conclusions：In both culture conditions,the totipotency of human embryonic stem cells is reserved perfectly.Comparatively,mTESR1culture system is much simpler,also,the morphology of single cell in mTESR1culture system is better.In all,mTESR1 system is an effective and optimized system for culturing stem cells.

关 键 词：人胚胎干细胞 饲养层细胞 mTESR1 MATRIGEL 

分 类 号：Q2-33[生物学—细胞生物学]