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1. 1江苏省农业科学院畜牧研究所 /农业农村部种养结合重点实验室， 南京 210014；2 山东奥克斯畜牧种业有限公司，济南 250100；3 香港大学李嘉诚医学院，香港 999077；4江苏优源奶业产业研究院有限公司，南京 211100
• 发布日期:2022-10-12

### Effects of Pladienolide B on Expression of Pluripotency Related Genes and Cell Viability of Bovine Embryonic Stem Cells

ZHAO Fang1, DING Qiang1, XIA ShuWen1, GAO YunDong2, LAN GuoCheng3, LIN ZhiPing4, WANG HuiLi1*, ZHONG JiFeng1* #br#

1. 1 Institute of Animal Science /Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture and Rural Affairs, Jiangsu Academy of Agricultural Sciences, Nanjing 210014; 2 Shandong OX Livestock Breeding Co.,Ltd, Jinan 250100; 3 Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong; 4 Jiangsu Youyuan Dairy Industry Research Institute Co. LTD, Nanjing 211100
• Online:2022-10-12

Abstract: 【Background Due to their high pluripotency, bovine embryonic stem cells (BESCs) have important application values in cattle breed conservation, breed selection and studying the regulation mechanism of livestock embryo development. However, studies on the maintenance of pluripotency and differentiation of BESCs are limited, the regulative mechanism remain unclear. 【Objective】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 which will provide 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】1. RT-qPCR results showed that the mRNA expression levels of SF3B1 and SF3B2 in EPSCM-BESCs were significantly down-regulated by 0.5nM to 1.5nM PlaB; When the PlaB concentration was 1.5nM, the mRNA expression of SF3B1 and SF3B2 in CTFR-BESCs were decreased; When the PlaB concentration ranged from 0.5nM to 1.5nM, 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. While PlaB concentration ranged from 0.5nM to 1.5nM, the mRNA expression of spliceosome LSM4 both in EPSCM-BESCs and CTFR-BESCs were significantly down-regulated. The concentration from 0.5nM to 1.5nM 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 1nM and 1.5nM PlaB while PlaB concentration from 0.5 to 1.5nM, 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 1.5nM, 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. And CTFR-BESCs was more sensitive than EPSCM-BESCs. 【Conclusion】PlaB significantly up-regulated the expression of pluripotent markers and partially totipotent markers in CTFR-BESCs and EPSCM-BESCs. 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 BESCs were not completely consistent. Due to the inhibiting effect of PlaB on cell viability of BESCs, further studies are needed to optimize the culture system.