›› 2010, Vol. 41 ›› Issue (1): 22-26.doi: 10.3969/j.issn.0529-1356.2010.01.005

• 论著 • Previous Articles     Next Articles

Mechanism of cyclin-dependent inhibitor p27Kip1 in regulating the differentiation of immortalized human neural progenitor cells

  

  1. 1. Key Laboratory of Neurodegenerative Diseases, Ministry of Education, Xuan Wu Hospital, Capital Medical University, Beijing100053, China;2. Department of Cell Biology, Capital Medical University, Beijing100069, China;3.Beijing Institute for Neuroscience, Capital Medical University, Beijing100069, China
  • Received:2008-11-03 Revised:2008-12-12 Online:2010-02-06
  • Contact: ZHANG Hai-yan

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

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