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通过在人胚胎干细胞中过表达转录因子FOXN1(forkhead box protein N1),观察细胞形态变化并检测标志基因的表达,成功地将其定向分化为胸腺上皮细胞。利用TALEN介导的同源重组在人胚胎干细胞中敲入FOXN1-m Cherry报告元件,使其能够指示内源FOXN1的表达。通过Teton诱导表达慢病毒系统在人胚胎干细胞系X-01中过表达FOXN1,发现人胚胎干细胞逐渐分化,细胞形态变扁平,细胞体积变大,表现出类似胸腺上皮细胞的形态。RT-PCR结果表明,过表达FOXN1后胸腺上皮细胞的标志基因TBX1、HOXA3、EYA1、K5、K8和内源FOXN1的m RNA水平都显著上调,并且随着诱导时间的延长而增加,在第12 d达到最高。对诱导后第12 d所得的细胞进行免疫染色可观察到K5(keratin 5)和K8(keratin 8)的表达,其中19%的细胞表达K5,45%的细胞表达K8。上述结果表明,通过过表达FOXN1可诱导人胚胎干细胞定向分化为胸腺上皮细胞。
Through the overexpression of FOXN1 (forkhead box protein N1) in human embryonic stem cells, the morphological changes of the cells were observed and the expression of the marker gene was detected, and it was successfully differentiated into thymus epithelial cells. FOXN1-m Cherry reporter elements were knocked in human embryonic stem cells using TALEN-mediated homologous recombination to enable them to indicate the expression of endogenous FOXN1. Expression of FOXN1 by Teton-induced lentivirus system in human embryonic stem cell line X-01, found that human embryonic stem cells gradually differentiated, the cell morphology became flat, the cell volume became larger, showing a similar thymus epithelial morphology. The result of RT-PCR showed that the levels of m RNA in the markers of the thymus epithelial cells overexpressing FOXN1 were significantly up-regulated after TBX1, HOXA3, EYA1, K5, K8 and endogenous FOXN1, and increased with the prolongation of induction time. d reached the highest. K5 (keratin 5) and K8 (keratin 8) expression were observed by immunostaining cells on day 12 after induction, with 19% of cells expressing K5 and 45% of cells expressing K8. The above results indicate that human embryonic stem cells can be induced to differentiate into thymocytes by overexpression of FOXN1.