关键词: 牛胚胎干细胞, Pladienolide B, 剪接体, 多能性相关基因, 细胞活力

Abstract:

【Background】 Due to high pluripotency of bovine embryonic stem cells (BESCs), they have important application values in cattle breed conservation, breed selection, and regulation mechanism study of livestock embryo development. However, the studies on the maintenance of pluripotency and differentiation of BESCs are limited, and the regulative mechanism remains unclear. 【Objective】The aim of this study was to investigate the effects of different concentrations of Pladienolide B (PlaB) on the expression of pluripotent markers, totipotent markers and embryonic cell-lineage genes as well as the cell viability of BESCs, so as to provide the reference and theoretical basis for improving the developmental potency of BESCs.【Method】Immunofluorescence was used to detect the expression of pluripotent markers of bovine BESCs, and real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the effects of different concentrations of PlaB on the expression of spliceosome, totipotent markers and embryonic cell-lineage genes of BESCs. RT-qPCR and Western Blot were used to detect the effects of different concentrations of PlaB on both mRNA and protein expression of pluripotent markers of BESCs. CCK8 and EDU staining was used to detect the effects of different concentrations of PlaB on the proliferation of BESCs. 【Result】 RT-qPCR results showed that the mRNA expression levels of SF3B1 and SF3B2 in EPSCM-BESCs were significantly down-regulated by 0.5 nmol·L^-1 to 1.5 nmol·L^-1 PlaB; when the PlaB concentration was 1.5 nmol·L^-1, the mRNA expressions of SF3B1 and SF3B2 in CTFR-BESCs were decreased; when the PlaB concentration ranged from 0.5 nmol·L^-1 to 1.5 nmol·L^-1, the mRNA expression levels of SF3B4 and SF3B5 in both CTFR-BESCs and EPSCM-BESCs were increased in a dose-dependent manner. Furthermore, PlaB significantly down-regulated mRNA expression of SF3B6 in the CTFR-BESCs. When PlaB concentration ranged from 0.5 nmol·L^-1 to 1.5 nmol·L^-1, the mRNA expression of spliceosome LSM4 both in EPSCM-BESCs and CTFR-BESCs were significantly down-regulated. The concentration from 0.5 nmol·L^-1 to 1.5 nmol·L^-1 PlaB significantly down-regulated the expression levels of EFTUD2 mRNA in CTFR-BESCs; the mRNA expression of EFTUD2 mRNA was significantly down-regulated in BEPSCM-BESCs with 1 nmol·L^-1 and 1.5 nmol·L^-1 PlaB while PlaB concentration from 0.5 to 1.5 nmol·L^-1, both the mRNA expression and protein levels of the pluripotent markers OCT4, SOX2 and NANOG in CTFR-BESCs and EPSCM-BESCs were up-regulated in a dose-dependent manner. By the concentration range from 0.5 to 1.5 nmol·L^-1, PlaB dose-dependently up-regulated the mRNA levels of totipotent markers such as MDM2, PID1 and BTG2 in CTFR-BESCs and EPSCM-BESCs, while the mRNA levels of DDIT4 and PDRG1 were down-regulated. The mRNA expression of embryonic cell lineage genes in the CTFR-BESCs were up-regulated while the PlaB was added. The addition of PlaB in EPSCM-BESCs significantly reduced the expression of GATA4, GATA6, SOX7 and other embryonic cell lineage genes, but had no significant effect on ZIC1. The cell viability of CTFR-BESCs and EPSCM-BESCs showed a downward trend with increasing of PlaB dose and treatment time, while CTFR-BESCs was more sensitive than EPSCM-BESCs. 【Conclusion】PlaB significantly up-regulated the expression of pluripotent markers and partial totipotent markers in CTFR-BESCs and EPSCM-BESCs, and the expression of gene lineages and cell viability in EPSCM-BESCs were decreased. The effective concentration and effects on gene expression of PlaB in the two types BESCs were not completely consistent. Due to the inhibiting effect of PlaB on cell viability of BESCs, the further studies were needed to optimize the culture system.

Key words: bovine embryonic stem cells, pladienolide B, spliceosome, pluripotency related genes, cell viability