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目的探讨全反式视黄酸(all-trans retinoic acid,atRA)诱导小鼠胚胎干细胞(mouse embryonicstem cells,mESCs)向原始生殖细胞(Primordial germ cell s,PGCs)分化的相关基因表达改变及其机制。方法 mESCs分化形成拟胚体(embryoid bodies,EBs),不同浓度atRA(1 μM,2 μM,5μM)持续诱导EBs 16h、2d和5d,观察EBs形态变化,实时PCR和Western blot检测PGCs分化相关基因和蛋白表达变化。分析基因启动子中RA反应元件,以Stra8(Stimulated by Retinoic Acid Gene 8,Stra8)基因为阳性对照,确定各基因对atRA刺激的原发性反应和继发性反应。结果不同浓度atRA诱导EBs 16h和2d后形态无明显变化。诱导后EBs的PGCs分化相关基因mRNA表达分4种情况:16h内(Stra8)表达量达最大,然后下降;2d内(Scp3)表达量达最大,然后下降;5d达到最大量(Mvh);表达量无明显变化(Fragilis、Blimp-1和Prm1)。Mvh表达符合继发反应特点;Stra8和Scp3表达符合原发反应特点;Blimp1、Prm1和Fragilis表达可能与atRA调节无关。Stra8蛋白变化与mRNA变化相一致,而Mvh则不一致。结论 atRA诱导mESCs向PGCs分化,各阶段标志基因表达变化均在相对短的时间内完成(2～5d),atRA诱导浓度可选用1 μM。根据基因表达模式初步得出,atRA调控的靶基因为Mvh,具备继发反应特点,与特异性分化有关,而Blimp-1、Fragilis和Prm1调控PGCs分化可能与atRA无关。
OBJECTIVE: To investigate the changes of gene expression related to differentiation of mouse embryonic stem cells (mESCs) into primordial germ cells (PGCs) induced by all-trans retinoic acid (atRA) and its mechanism . Methods EBs were differentiated into mESCs. EBs were continuously induced at different concentrations of atRA (1 μM, 2 μM, 5 μM) for 16 h, 2 d and 5 d. The morphological changes of EBs were observed. Real-time PCR and Western blot were used to detect the expression of PGCs And protein expression changes. The RA response element in the gene promoter was analyzed. Stra8 (Stimulated by Retinoic Acid Gene 8, Stra8) gene was used as a positive control to determine the primary and secondary reaction of each gene to atRA stimulation. Results There was no significant change in the morphology of EBs induced by atRA at different concentrations for 16 h and 2 d. The mRNA expression of PGCs in EBs induced by EBs was divided into four groups: Stra8 expression reached the maximum within 16 hours and then decreased. Scp3 expression reached its maximum at 2 days and then decreased, and reached the maximum at 5 days (Mvh) There was no significant change in the amount (Fragilis, Blimp-1 and Prm1). Mvh expression conforms to the secondary reaction characteristics; Stra8 and Scp3 expression consistent with the primary reaction characteristics; Blimp1, Prm1 and Fragilis expression may be independent of atRA regulation. Stra8 protein changes consistent with mRNA changes, while Mvh is inconsistent. Conclusions AtRA induces the differentiation of mESCs into PGCs. The expression of the marker genes in each stage is completed in a relatively short period of time (2 ~ 5 days), and the concentration of atRA is 1 μM. According to the gene expression pattern, the target gene regulated by atRA is Mvh, which has the characteristics of secondary reaction, which is related to specific differentiation. However, the regulation of PGCs by Blimp-1, Fragilis and Prm1 may not be related to atRA.