昆明小鼠胚胎干细胞建系的初步研究  

Primary study on establishment of embryonic stem cells from Kunming mouse

作　　者：戢开丽[1] 仇恒滨[1] 严为巧[1] 庄新杰[1] 段亚苹[1] 张明[1] 卢盛晟[1] 卢克焕[1] 

机构地区：[1]广西大学动物繁殖研究所,广西南宁530005

出　　处：《广西农业生物科学》2008年第4期329-334,共6页Journal of Guangxi Agricultural and Biological Science

基　　金：广西自然科学基金项目(桂科自0640019)

摘　　要：以昆明小鼠(Mus musculus)为材料,利用超数排卵获得小鼠扩展囊胚、孵化囊胚。为了研究小鼠胚胎干细胞(Mouse Embryonic Stem Cells,mESC)分离和传代的影响因素,设计两个实验:(1)不同孕期(3.5 d或4.5 d)的囊胚对胚胎干细胞分离效率的影响;(2)第5代后,不同传代方法(机械法或酶消化法)对mESC传代的影响。结果显示:在3.5 d孕期时,胚胎为扩展囊胚,在4.5 d时为孵化囊胚,扩展囊胚在贴壁率、原代克隆形成率差异不显著,从在继代培养上显著高于孵化囊胚(P<0.05);第5代后用机械法或胰酶消化法传代对mESC的维持没有明显差别,但从克隆的形态上看,酶消化法优于机械法。最后,本研究从扩展囊胚组中成功获得了一株mESC,该mESC碱性磷酸酶染色呈强阳性,经RT-PCR分析显示表达Oct4、Sox2,核型为正常40XY。In the study, the expanded and hatched blastocysts were used, which were obtained from Kunming mouses by superovulation, to observe the factors affecting the isolation and subculture of the mouse embryonic stem cells （mESC）. Two experiments were designed. Experiment one focused on the effect of pregnancy states （3.5 d or 4. 5 d） on mESC isolation and subculture; Experiment two focused on the effect of passaging methods （mechanical or enzymatic method） on the mESC subculture after passage 5. The results showed that the 3. 5 d blastocystes were expanded whereas the 4. 5 d blastocysts were hatched. The difference between the expanded and hatched blastocysts was not significant at attachment and primary colonies formation rate, but the subculture of mESC from expanded blastocysts were better than the mESC from hatched blastocysts （P〈0.05）. After the fifth passage, judged from the morphology, the enzymatic method was significant better than the mechanical method. Finally, from the expanded blastocysts, a strain of mESC was successfully derived. This mESC strain had general mESC characteristics., normal mESC morphology, Alkaline phosphatase positive, expressing Oct4 and Sox2, and normal karyotype 40XY.

关 键 词：昆明小鼠 囊胚 胚胎干细胞 机械法 酶消化法 

分 类 号：Q813[生物学—生物工程]