Pladienolide B对牛胚胎干细胞多能性相关基因表达及细胞活力的影响  

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

作　　者：赵芳[1] 丁强 夏淑雯 高运东 兰国成 林志平 王慧利[1] 仲跻峰 ZHAO Fang;DING Qiang;XIA ShuWen;GAO YunDong;LAN GuoCheng;LIN ZhiPing;WANG HuiLi;ZHONG JiFeng(Institute of Animal Science,Jiangsu Academy of Agricultural Sciences/Jiangsu Province Engineering Research Center for Precision Animal Breeding/Key Laboratory of Crop and Animal Integrated Farming,Ministry of Agriculture and Rural Affairs,Nanjing 210014;Shandong OX Livestock Breeding Co.,Ltd,Jinan 250100;Li Ka Shing Faculty of Medicine,The University of Hong Kong,Hong Kong 999077,china;Jiangsu Youyuan Dairy Industry Research Institute Co.Ltd,Nanjing 211100)

机构地区：[1]江苏省农业科学院畜牧研究所/江苏省畜禽精准育种工程研究中心/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,中国香港999077 [4]江苏优源奶业产业研究院有限公司,南京211100

出　　处：《中国农业科学》2023年第11期2186-2201,共16页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2021YFD1200404);江苏省自然科学基金面上项目(BK20221433);江苏省农业科学院探索性颠覆性创新计划项目(ZX(21)1214);江苏省农业科技自主创新资金(CX(19)2037)。

摘　　要：【背景】牛胚胎干细胞(bovine embryonic stem cells,BESCs)因具备较高的多能性,在牛品种保存、品种选育及研究家畜胚胎发育调控机制方面具有重要的应用价值。然而,BESCs多能性维持及分化调控的研究相对缺乏,很多机制仍不清楚。【目的】探究不同浓度Pladienolide B(PlaB)对BESCs多能标记基因、全能标记基因、胚胎细胞谱系基因表达及细胞活力的影响,为提高BESCs多能性提供参考和理论依据。【方法】利用免疫荧光检测牛BESCs多能标记基因的表达,利用实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)检测不同浓度PlaB对BESCs剪接体、全能标记基因及胚胎细胞谱系基因表达的影响,利用RT-qPCR和Western Blot分别检测不同浓度PlaB对BESCs多能标记基因mRNA和蛋白表达的影响,利用CCK8和EDU染色检测不同浓度PlaB对BESCs增殖的影响。【结果】RT-qPCR结果显示,PlaB浓度为0.5—1.5 nmol·L^(-1)时显著下调EPSCM-BESCs中SF3B1和SF3B2的mRNA表达水平;PlaB浓度为1.5nmol·L^(-1)时,CTFR-BESCs中SF3B1和SF3B2的mRNA表达下降;PlaB浓度范围为0.5—1.5 nmol·L^(-1)时,CTFR-BESCs和EPSCM-BESCs的SF3B4和SF3B5的mRNA表达水平呈剂量依赖性增加。此外,PlaB显著下调CTFR-BESCs中SF3B6的mRNA表达。PlaB浓度为0.5—1.5 nmol·L^(-1)时,EPSCM-BESCs和CTFR-BESCs中剪接体LSM4的mRNA表达显著下调。PlaB浓度为0.5—1.5 nmol·L^(-1)时,显著下调CTFR-BESCs的EFTUD2 mRNA表达水平;PlaB浓度为1—1.5 nmol·L^(-1)时能显著下调BEPSCM-BESCs的EFTUD2 mRNA表达水平。浓度为0.5—1.5 nmol·L^(-1)范围内,PlaB均以剂量依赖性上调CTFR-BESCs和EPSCM-BESCs中多能标记基因OCT4、SOX2及NANOG的mRNA表达水平和蛋白水平。在PlaB浓度为0.5—1.5 nmol·L^(-1)范围内,PlaB以剂量依赖性上调CTFR-BESCs和EPSCM-BESCs中全能标记基因MDM2、PID1及BTG2的mRNA表达水平,而下调DDIT4和PDRG1的mRNA表达水平。CTFR-BESCs中添加PlaB均上调胚胎细胞谱系基因�【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-BE

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 

分 类 号：S823.2[农业科学—畜牧学]