bFGF培养体系中添加不同因子对绵羊类胚胎干细胞多能性的影响

Effect of bFGF culture medium complemented with different factors on pluripotency of ovine embryonic stem-like cell

为筛选出能够维持绵羊类胚胎干细胞(oESC-like)多能性的因子,以oESC-like为材料,在N2B27/CH/bFGF培养体系中,分别加入PD、PD/hLIF、PD/BPM4、PD/hLIF/BMP4,培养细胞8 d,分析绵羊类胚胎干细胞多能性,确定最适培养体系。结果显示:在N2B27/CH/bFGF培养体系添加PD或PD/hLIF后,oESC-like细胞生长较好;所有培养液中oESC-like细胞的AKP染色及多能性候选基因Sox2和Oct4免疫荧光蛋白染色结果均呈阳性;添加PD后,多能性候选基因Oct4与Klf4 mRNA的表达量显著高于N2B27/CH/bFGF培养体系中的(P<0.01),Nestin与Lin28的表达量显著降低(P<0.01),Sox2与c-Myc升高但差异不显著。添加PD/hLIF后,Oct4的表达量显著高于N2B27/CH/bFGF培养体系中的(P<0.01),c-Myc、Klf4、Lin28表达量升高但不显著,Nestin和Sox2表达量降低但也不显著。以上结果表明:在oESC-like培养体系中,添加PD或PD/hLIF可能有利于维持绵羊类胚胎干细胞的多能性。

To select the most effective growth factor maintaining the pluripotency of ovine embryonic stem-like cell （oESC-like）, PD, PD/hLIF, PD/BPM4 and PD/hLIF/BMP4 were added respectively into the culture system N2B27! CH/bFGF. After growing for 8 d, oESC-like cells were collected to determine the expression levels of pluripotency candi-date genes. When added with PD or PD/hLIF, oESC-like cell grew well. Staining results showed that oESC-like cells and Sox2 and Oct4 cultured in all media were positive. Compared with N2B27/CH/bFGF, mRNA levels of Oct4 and Klf4 in culture solution added with PD significantly increased （P〈 0.01 ）, while mRNA levels of Nestin and Lin28 decreased, and Sox2 and c-Myc did not remarkably in-crease, mRNA level of Oct4 in culture solution added with PD/hLIF significantly increased （P〈0. 01 ）. It was con- cluded that adding PD or PD/hLIF in N2B27/CH/bFGF system might be more favorable to maintain pluripotency of oESC-like cells.