关键词: p27Kip1, 细胞分化, S期激酶相关蛋白2, 细胞培养, 流式细胞术, 免疫印迹法, 永生化人神经前体细胞

Abstract: Objective To investigate whether there is any functional link between p27Kip1 function and all-trans retinoic acid (RA) in the control of neuronal differentiation of immortalized human neural progenitor cells (hSN12W-TERT cells). To investigate the mechanism by which p27Kip1 regulates the differentiation of immortalized human neural progenitor cells. Methods hSN12W-TERT cells were derived from the striatums of human embryos at 12 weeks gestation and cultured with serum-free medium in presence of EGF and bFGF. At the appropriated time, hSN12W-TERT cells were exposed to 1μmol/L RA for 3, 5, 7 days respectively. The experiment was repeated there times. Cell cycle analysis was performed by flow cytometry analysis (FACS). The expression of p27Kip1, p21Cip1, cyclin-dependent kinase 2 (cdk2）, p-cdk2 and S-phase kinase-associated protein 2 (skp2) in hSN12W-TERT cells before and after RA treatment cells were determined by using Western blotting analysis. Results FACS result showed that 77.25% of proliferating hSN12W-TERT cells were in the G1/G0-phase while 9.38% of cells in the S-phase. Following RA treatment, cell growth was arrested, and 85.68% of cells accumulated in G1/G0-phase while 8.57% of cells in the S-phase. Western blotting analysis demonstrated that the levels of p27SUP>Kip1/SUP> in the hSN12W-TERT cells increased following 3 days’ treatment with RA compared with those of normal untreated cells, with a peak at 5 days (EM>P/EM><0.05). The similar results were acquired both in nuclear proteins and in cytoplasm proteins of hSN12W-TERT cells. The expression level of p21SUP>Cip1/SUP> decreased 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. Skp2, which was required for the ubiquitin-mediated degradation of p27SUP>Kip1/SUP>, was detected in proliferating hSN12W-TERT cells. The expression of skp2 reduced dramatically in response to RA treatment in a time-dependent manner.Conclusion There is a functional link between RA and p27SUP>Kip1/SUP> function in the control of neuronal differentiation in hSN12W-TERT cells. p27SUP>Kip1/SUP> plays a key role during neuronal differentiation. Moreover, high levels of p27SUP>Kip1/SUP> are associated with its degradation inhibiting through reducing proteasome-dependent

Key words: p27SUP>Kip1/SUP>, Cell differentiation, Sphase kinaseassociated protein 2, Cell culture, Flow cytometry, Western blotting, Immortalized human neural progenitor cell