研究旨在探究差异代谢物泛酸(PA)对鸡胚胎干细胞(ESCs)向原始生殖细胞(PGCs)分化过程中的作用,为从代谢角度解析PGCs形成机制提供新的理论基础.试验基于转录组学筛选出PA,通过CCK8与EdU试验探究PA及其代谢抑制剂PZ的适宜添加浓度;最后,分别通过观察细胞形态、qRT-PCR检测PGCs特异标记基因、间接免疫荧光和流式细胞分析等分析PA代谢在ESCs向PGCs分化过程中的作用.结果显示:参与PA代谢相关的基因在ESCs与PGCs中呈现差异性表达,且富集于生殖细胞分化相关的通路(Pantothenate and CoA biosynthesis、TGF-beta signaling pathway和AMPK signaling pathway);PA和PZ的适宜添加浓度分别为20 µmol/L和80 nmol/L;PA组在第6天的类胚体数目显著低于PZ组,PA组中CVH和C-KIT3的表达量显著低于PZ组(P<0.01);PA组的DDX4阳性细胞数显著低于PZ组(P<0.01).结果表明添加PA抑制PGCs的形成,添加PZ促进PGCs的形成.
Screening of the Metabolite Pantothenic Acid and Its Functional Verification in the Formation Process of Primordial Germ Cells
The purpose of this study was to explore the effect of the differential metabolite pantothenic acid(PA)on the dif-ferentiation process of chicken embryonic stem cells(ESCs)into primordial germ cells(PGCs),and to provide a new theoretical basis for analyzing the formation mechanism of PGCs from a metabolic perspective.The experiment screened out PA based on transcriptomics,and then used CCK8 and EdU experiments to explore the optimal concentration of PA and its metabolic inhibitor PZ.Finally,the cell morphology was observed,qRT-PCR was used to detect PGCs-specific marker genes,indirect immunofluo-rescence and Flow cytometry analysis was used to analyze the role of PA metabolism in the differentiation process of ESCs into PGCs.The results showed that genes involved in PA metabolism were differentially expressed in ESCs and PGCs,and were en-riched in pathways related to germ cell differentiation(Pantothenate and CoA biosynthesis,TGF-beta signaling pathway and AMPK signaling pathway).The optimal addition concentrations of PA and PZ were 20 µmol/L and 80 nmol/L,respectively.The number of embryoid bodies in the PA group on the 6th day was significantly lower than that in the PZ group;The expression levels of CVH and C-KIT3 in the PA group were significantly lower than those in the PZ group(P<0.01).The number of DDX4-positive cells in the PA group was significantly lower than that in the PZ group(P<0.01).The above indicated show that the addition of PA inhibits the formation of PGCs.
万方数据
维普
