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首页> 美国卫生研究院文献>Bioscience Reports >Dynamic expression and regulatory mechanism of TGF-β signaling in chicken embryonic stem cells differentiating into spermatogonial stem cells
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Dynamic expression and regulatory mechanism of TGF-β signaling in chicken embryonic stem cells differentiating into spermatogonial stem cells

机译：TGF-β信号在鸡胚胎干细胞分化为精原干细胞中的动态表达及调控机制

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The present study investigated the dynamic expression and regulatory mechanism of transforming growth factor β (TGF-β) signaling involved in embryonic stem cells (ESCs) differentiation into male germ cells. Candidate genes involved in TGF-β signaling pathway were screened from RNA-sequencing (RNA-seq), which were further validated by quantitative real-time PCR (qRT-PCR). Bone morphogenetic protein 4 (BMP4) was used to induce differentiation of ESCs in vitro. Inhibition of TGF-β signaling pathway was reflected by Western blot of SMAD2 and SMAD5 expression. Differentiating efficiency of germ cells was evaluated by immunofluorescence and fluorescence-activated cell sorting (FACS). Germ cell marker genes were assessed by qRT-PCR in the differentiation process, with activation or inhibition of TGF-β signaling pathway. In the process of in vitro induction, SMAD2 and SMAD5 were found to significantly up-regulated in BMP4 group versus the control and inhibition groups after 4 and 14 days. Expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6 were significantly increased in the BMP4 group compared with the control group, while down-regulated in the inhibition groups. The proportion of germ cell-like cells was decreased from 17.9% to 2.2% after 4 days induction, and further decreased from 14.1% to 2.1% after 14 days induction. Correspondingly, expression of marker genes in germ cells was significantly lower. In vivo inhibition of TGF-β signaling pathway reduced germ cells formation from 5.5% to 1.6%, and down-regulated the expression of CKIT, CVH, DAZL, STRA8, and INTEGRIN α6. In conclusion, our study reveals the mechanism regulating spermatogonial stem cells (SSCs) and lays the basis for further understanding of the regulatory network.
机译：本研究调查了涉及胚胎干细胞（ESC）分化为雄性生殖细胞的转化生长因子β（TGF-β）信号转导的动态表达和调控机制。从RNA测序（RNA-seq）中筛选了参与TGF-β信号通路的候选基因，并通过实时定量PCR（qRT-PCR）对其进行了进一步验证。骨形态发生蛋白4（BMP4）用于体外诱导ESC分化。 TGF-β信号通路的抑制作用通过SMAD2和SMAD5表达的蛋白质印迹来反映。通过免疫荧光和荧光激活细胞分选（FACS）评估生殖细胞的分化效率。在分化过程中，通过激活或抑制TGF-β信号通路，通过qRT-PCR评估生殖细胞标记基因。在体外诱导过程中，在第4天和第14天后，与对照组和抑制组相比，BMP4组的SMAD2和SMAD5明显上调。与对照组相比，BMP4组的CKIT，CVH，DAZL，STRA8和INTEGRINα6的表达显着增加，而在抑制组中则下调。诱导4天后，生殖细胞样细胞的比例从17.9％降至2.2％，诱导14天后从14.1％进一步降至2.1％。相应地，标记基因在生殖细胞中的表达明显降低。体内对TGF-β信号通路的抑制将生殖细胞的形成从5.5％降低到1.6％，并下调CKIT，CVH，DAZL，STRA8和INTEGRINα6的表达。总之，我们的研究揭示了调节精原干细胞（SSCs）的机制，并为进一步了解调节网络奠定了基础。

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