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目的研究p27Kip1在永生化人神经前体细胞分化中的作用,探讨神经前体细胞的分化机制。方法取本课题组已建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)进行培养,在细胞进入对数生长期时给予1μmol/L全反式视黄酸(RA)诱导,重复诱导3次,每次均在诱导的第3、5、7天收集细胞,用流式细胞仪分析RA诱导第3天细胞周期的变化,用免疫印迹法检测RA诱导第3、5、7天p27Kip1、p21cip1细胞周期蛋白依赖激酶2(cdk2)及S期激酶相关蛋白2(skp2)的变化。结果流式细胞术结果显示,hSN12W-TERT细胞经1μmol/LRA诱导3d后,G0/G1期细胞的比例由77.25%上升至85.68%,而S期的比例由9.38%下降到8.57%。免疫印迹法结果显示,RA诱导第3天,hSN12W-TERT细胞p27Kip1蛋白表达比未经RA诱导的细胞增加,并在RA诱导第5天达到高峰(P<0.05)。未经RA诱导的正常hSN12W-TERT细胞p21Cip1蛋白表达弱,RA诱导后p21Cip1蛋白的表达略呈下降趋势。RA诱导前后hSN12W-TERT细胞cdk2蛋白的表达变化不明显,但反映cdk2活性的磷酸化cdk2(p-cdk2)的表达在RA诱导后明显下降,同时,参与p27Kip1泛素降解途径的重要因子skp2的表达在RA诱导后明显下降。结论在RA诱导hSN12W-TERT细胞分化的过程中,p27Kip1通过抑制cdk2的活性而发挥促细胞分化的作用,且RA诱导后p27Kip1蛋白含量增加与其泛素降解途径被抑制密切相关。
Objective To study the role of p27Kip1 in the differentiation of immortalized human neural progenitor cells and to explore the differentiation mechanism of neural progenitor cells. Methods The immortalized human neural progenitor cell line hSN12W-TERT cells (passage 12) established in this research group were cultured and induced by 1 μmol / L all-trans retinoic acid (RA) when the cells entered the logarithmic growth phase , 3 times repeated induction, each collected on the induction of 3,5,7 days cells were analyzed by flow cytometry RA induced cell cycle changes on the 3rd day, Western blot was used to detect the induction of RA 3, 7 days p27Kip1, p21cip1 cyclin-dependent kinase 2 (cdk2) and S-kinase 2 (skp2) changes. Results The results of flow cytometry showed that the percentage of cells in G0 / G1 phase increased from 77.25% to 85.68% in hSN12W-TERT cells after being treated with 1μmol / LRA for 3 days, while the proportion of S phase decreased from 9.38% to 8.57%. Western blotting showed that the expression of p27Kip1 protein in hSN12W-TERT cells was increased on the 3rd day after RA induction than that in the non-RA induced cells, and peaked on the fifth day after RA induction (P <0.05). The expression of p21Cip1 protein in normal hSN12W-TERT cells without RA induction was weak, and the expression of p21Cip1 protein was slightly decreased after RA induction. The expression of cdk2 protein in hSN12W-TERT cells did not change significantly before and after RA induction, but the expression of phosphorylated cdk2 (p-cdk2), which reflects the activity of cdk2, was significantly decreased after RA induction. Meanwhile, the expression of cdk2, an important factor involved in the ubiquitin degradation pathway of p27Kip1, Expression was significantly decreased after RA induction. Conclusion p27Kip1 plays a role in promoting differentiation of hSN12W-TERT cells by inhibiting the activity of cdk2 during the differentiation of hSN12W-TERT cells induced by RA. The increase of p27Kip1 protein after RA induction is closely related to the inhibition of its ubiquitin degradation pathway.