KONG Huijuan,SUN Yingpu,GENG Jiaxuan.Influencing factors in the establishment of mouse embryonic stem cells[J].JOURNAL OF ZHENGZHOU UNIVERSITY(MEDICAL SCIENCES),2012,(01):36.





Influencing factors in the establishment of mouse embryonic stem cells
郑州大学第一附属医院生殖医学中心 郑州450052
KONG HuijuanSUN YingpuGENG Jiaxuan
Medical Reproductive Center,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052
mouseembryonic stem cellculture mediuminner cell mass
目的:探讨昆明小鼠胚胎干细胞(ESC)分离后克隆形成和细胞传代的几种影响因素,对其特性作初步鉴定。方法:对比超排卵和自然受孕2种囊胚获取方法、囊胚培养液和高糖DMEM培养液2种培养液对其克隆形成和细胞传代的影响。对贴壁后形成的原代ESC克隆进行评分。稳定传代的一株ESC进行鉴定。结果:超排卵组、自然受孕组的内细胞团形成率(64.9% vs 68.2%)和原代ECS克隆形成率(10.1% vs 14.5%)比较,差异均无统计学意义(χ2=0.345和1.385,P=0.557和0.239)。囊胚培养液组24 h囊胚孵出率明显高于DMEM培养液组(56.8% vs 17.1%)(χ2=33.026,P=0.001),原代ESC克隆形成率明显低于DMEM培养液组(35.2% vs 15.9%)(χ2=9.021,P=0.002)。ICM的评分与ESC克隆传代能力相关(r=0.531,P<0.001),直径70~100 μm、周边规则、隆起明显的ICM,ESC克隆传代持久。结论:高糖DMEM培养液对克隆的分离和传代有利。对ICM贴壁后形成的ESC克隆进行评分有助于确定挑选克隆传代的时机。
Aim:To investigate several kinds of influencing factors of Kuming mice embryonic stem cells(ESC) isolation and to perform the preliminary identification.Methods:Two blastocystobtaining methods,and two kinds of mediums (HLM and DLM) were compared, respectively. And the inner cell mass(ICM) was scored. Moreover, the omnipotent identification was carried out in a ESC strain with stable passage.Results:The ICM formation rates of the superovulation group and natural conception group (64.9% vs 68.2%) as well as the colony formation rate of the primary generation ESC (10.1% vs 14.5%) were similar, with no significant differences(χ2=0.345 and 1.385,P=0.557 and 0.239). The 24hour hatching rate of the HLM group was significantly higher than the DLM group(56.8% vs 17.1%)(χ2=33.026,P=0.001). But the colony formation rate of the primary generation was significantly lower (35.2% vs 15.9%)(χ2=9.021,P=0.002).Scoring of ICM was related to the ability of ESC clones passaged(rs=0.531,P<0.001).The ICM with a diameter of 70~100 μm, regular boundary and obvious elevation could have more passages.Conclusion:The high glucose DMEM medium is beneficial to the isolation and passage of cell clones. Scoring of the ICM is helpful to determine the timing of selecting clones and passage.


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*国家自然科学基金资助项目30771105 doi:10.3969/j.issn.16716825.2012.01.012 #通讯作者,女,1962年1月生,博士,主任医师,教授,研究方向:生殖医学,Email:syp2008@vip.sina.com
更新日期/Last Update: 1900-01-01