人神经前体细胞分化过程中p27^(Kip1)的作用及其调节机制研究

Retinoic acid induces differentiation of human neural progenitor cells by cyclin-dependent inhibitor p27^(Kip1)

目的研究视黄酸(RA)诱导人神经前体细胞(hNPCs)分化的过程中,细胞周期调节蛋白p27Kip1的变化及其调节机制,以掌握hNPCs的增殖分化特性。方法取原代hNPCs体外培养,在细胞进入对数生长期时给予RA诱导,在诱导d3、5、7收集细胞,用流式细胞分析术观察细胞周期的变化、用Westernblot法观察p27Kip1及其相关的细胞周期调节蛋白p21Cip1、cdk2的变化,以及参与p27Kip1泛素-蛋白酶降解途径的重要因子skp2的变化。结果RA诱导d3,hNPCs细胞G0/G1期的比例由诱导前的65.20%上升至80.72%;而S期的比例由诱导前的28.39%下降到8.62%;p27Kip1蛋白表达在RA诱导d3增加,并在d5达到高峰;p21Cip1和cdk2的表达在RA诱导前后变化不明显,但反映cdk2活性的p-cdk2蛋白的表达在RA诱导后明显下降;skp2的表达在RA诱导后明显下降。结论在RA诱导hNPCs分化的过程中,p27Kip1蛋白泛素降解途径受阻,致使其含量增加,并通过抑制cdk2的活性而发挥了促进hNPCs分化的作用。

Aim To investigate whether human neural progenitor cells （hNPCs） could be induced to differen- tiate toward a neuronal phenotype by all-trans Retinoic acid （RA） , and to study whether p27Kip1 played a key role in the differentiation process of hNPCs. Methods hNPCs were derived from the striatums of human embryos at 12 weeks＇ gestation and cultured with ser- um-free medium in presence of EGF and bFGF. At the appropriated time, hNPCs were exposed to 1 μmol· L-1 RA for 3,5,7days respectively. The properties of hNPCs were characterized by using flow cytometry a- nalysis（FACS） and Western blot. Results Cell cycle analysis was performed by FACS. Typically,65.20% of proliferating hNPCs were in the G1/G0-phase while 28.39% of cells in the S-phase. Following RA treat- ment, cell growth was arrested, and 85. 72% of cells accumulated in G1/G0-phase while 8.62% of cells in the S-phase. Western blot analysis demonstrated that the levels of p27Kip1 in the hNPCs increased following 3 days＇ treatment with RA compared with those of un- treated cells,with a peak at 5th days （P 〈0. 05）. The same results were acquired both in nuclear proteins and in cytoplasm proteins. The expression level of （s-phase kinase-associated protein 2, skp2 ） decreased signifi- cantly in response to RA treatment. RA did not affect the expression of cdk2, but the expression of p-cdk2 , which represented the activity of cdk2, was markedly decreased in response to RA treatment. Proliferating cell nuclear antigen （PCNA） could be detected only in normal untreated cells and disappeared when cells were induced to differentiate by RA. skp2, which was re- quired for the ubiquitin-mediated degradation of p27Kip1, was detected in proliferating hNPCs. The ex- pression of skp2 reduced dramatically in response to RA treatment in a time-dependent manner. Conclu- sions There was a functional link between RA and p27x^p~ function in the control of neuronal differentia- tion in hNPCs, p27Kip1 played a key role in neuronal differentiation. Moreover, high levels of p27Kip1 were regulated via inhibiting its degradation through reducing proteasome-dependent proteolysis.