利用单细胞融合技术高效获得融合细胞的方法

Method of efficiently obtaining the intraspecies fused cells by single cell fusion

试验以小鼠胚胎干细胞(mESC)作为细胞融合对象,用0.25%胰酶将mESC消化成单细胞,利用细胞凝集素构建一对一的单细胞对,在含有50%聚乙二醇的ES培养液中培养1 min,随后将细胞继续培养7 d,获得既表达红色荧光也表达绿色荧光的克隆即为融合成功。结果显示,单细胞融合法的克隆效率(46.25%)显著高于传统融合的效率(0.001 7%)。进一步检测融合细胞的生物学特性发现,虽然细胞形态与mESC具有很大的差异,但依然呈AP阳性,RNA水平上表达Oct4、Sox2和Nanog,具有向三胚层分化的能力等。结果表明,单细胞融合法是一种高效构建融合细胞的新途径。

Cell fusion is an important means to study cell pluripotency,fate determination and gene regulation.However,the efficiency of cell fusion methods currently reported is generally around 1%.In order to improve the efficiency of cell fusion,it is more effective to provide research materials for related research.This study proposed a single cell fusion method.Mouse embryonic stem cells(mESCs) were used as a cell fusion material,mESCs were digested with 0.25% trypsin into single cells,one to one pair was constructed with phytohemagglutinin,cultured in 50% polyethylene glycol for 1 min,then the cells were cultured for 7 days,got clones expressing both red and green fluorescence.The fusion efficiency of the single cell fusion method(46.25%) was significantly higher than that of the conventional fusion(0.001 7%).Throught further examination of the biological characteristics there was a great difference between the fusion cells,and mouse ES cell morphology,with AP positive;RNA expression of OCT4,SOX2 and Nanog and the ability to differentiate to the three germ layers and so on.The results indicate that the single cell fusion method is a new way to get fusion cells.