大鼠类胚体的制备及实时观察

Preparation and Real-time Observation of Rat Embryoid Bodies

目的探讨悬滴法与悬浮法制备的大鼠类胚体(embryoid bodies,EBs)的差异以及培养时间对EBs所包含三胚层组织特异性基因表达的影响。方法使用悬滴法与悬浮法将大鼠胚胎干细胞(embryonic stem cells,ESCs)制备为EBs并连续培养10 d,在第5天和第10天分别对两种方法制备的EBs进行形态学观察,并进行数量统计与直径检测,随后提取EBs蛋白与mRNA,通过Western blot与RT-qPCR实验检测EBs三胚层特异性蛋白和基因的表达水平。结果与悬浮法相比,悬滴法制备的EBs生成率低(P<0.01),但同质性更好;ESCs和EBs均表达多能基因Naong和OCT4,但随着培养时间的增长,多能基因表达逐渐降低,与ESCs相比,EBs(xf5、xf10和xd10)中OCT4与Naong基因的表达量均降低(P均<0.05)。EBs成功表达AFP、Gata-6、cdh1、Gata-1、Mixl1、Brachyury、Nestin以及VIM等三胚层特异性基因,且随着EBs培养天数的增加,内胚层基因AFP表达升高(P<0.05),Gata-6和cdh1的表达无明显变化(P>0.05);中胚层基因Brachyury表达降低(P<0.05),Gata-1和Mixl1表达无明显变化(P>0.05);外胚层基因VIM表达降低(P<0.05),Nestin表达无明显变化(P>0.05)。在同时期不同方法制备的EBs中,xf10的EBs三胚层特异性基因表达高于xd10的EBs(P<0.05)。结论悬滴法制备的EBs生成率低于悬浮法,但大小均一,分化同步,培养时间对EBs特异性基因的表达具有重要的影响。

Objective To investigate the difference of rat embryonic bodies prepared by hanging drop method and suspension method,and the effect of culture time on the expression of tissue-specific genes in the three germ layers of EBs.Methods Rat embryonic stem cells(ESCs)were prepared as EBs by hanging drop method and suspension method and cultured for 10 days.Quantitative statistics and diameter detection were performed,and then the protein and mRNA of EBs were extracted at 5 days and 10 days.Western blot and RT-qPCR experiments were used to detect the tissue-specific gene expression levels of the three germ layers contained in EBs.Results Compared with the suspension method,the production rate of EBs prepared by the hanging drop method was lower(P<0.01),but the homogeneity was better. Both ESCs and EBs expressed the pluripotent genes Naong and OCT4,but the expression of pluripotent genes gradually decreased with the increase of culture time. Compared with ESCs,the expression of OCT4 and Naong genes in EBs(xf5,xf10 and xd10) decreased (P all<0.05);EBs successfully expressed AFP,Gata-6,cdh1,Gata-1,Mixl1,Brachyury,Nestin,VIM and other three germ layer-specific genes,and with the increase of culture days of EBs,the expression of endoderm gene AFP increased(P<0.05),the expression of Gata-6 and cdh1 had no significant change(P>0.05);the expression of mesoderm gene Brachyury decreased(P<0.05),and the expression of Gata-1 and Mixl1 had no significant change(P>0.05);the expression of ectoderm gene VIM decreased (P<0.05),there was no significant change in Nestin expression(P>0.05);at the same period but different prepared methods in the EBs,the three germ layer-specific gene expressions of EBs suspended for 10 days were higher than those of EBs suspended for 10 days(P<0.05). Conclusion The production rate of EBs prepared by hanging drop method is significantly lower than that by suspension method,but the size is uniform and the differentiation is synchronous,and the culture time has an important effect on the expression of EBs specific genes.