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目的:利用全反式视黄酸(all-trans retinoic acid,RA)诱导永生化的人神经前体细胞(immortalized human neural progenitor cells,hSN12W-TERTcells)分化,研究分化过程中细胞周期调节蛋白p27Kip1,p21Cip1,细胞周期蛋白激酶2(cyclin-dependent kinase2,cdk2)及细胞周期蛋白E(cyclinE)的变化,探讨永生化人神经前体细胞分化的相关分子机制。方法:取本课题组已经建立的永生化人神经前体细胞系hSN12W-TERT细胞(第12代)培养并给予1μmol/L RA诱导。在RA诱导的第3,7d观察细胞形态变化,用RT-PCR方法检测RA诱导前后hSN12W-TERT细胞p27Kip1,p21Cip1,cdk2及cyclinE mRNA的变化,用免疫细胞化学染色方法比较RA诱导前后p27Kip1蛋白表达的变化。结果:RA诱导第3d,hSN12W-TERT细胞比未经RA诱导的正常hSN12W-TERT细胞生长缓慢且形态发生改变,表现为胞体变小,突起延长增多。至RA诱导第7d时,hSN12W-TERT细胞形态变化更加明显,接近成熟神经元形态。RT-PCR结果表明,hSN12W-TERT细胞中p27Kip1mRNA的表达在RA诱导后明显增加,而p21Cip1mRNA的表达在RA诱导后略呈下降趋势。hSN12W-TERT细胞中cdk2、cyclinE的mRNA水平在RA诱导前后没有明显变化。免疫细胞化学染色结果显示,RA诱导第3d,hSN12W-TERT细胞p27Kip1的表达比未经RA诱导的正常hSN12W-TERT细胞明显增加(P<0.05),RA诱导第7d,p27Kip1的表达进一步增加(P<0.05)。结论:p27Kip1参与RA诱导的永生化人神经前体细胞生长阻滞和分化过程,p27Kip1可能在永生化人神经前体细胞的神经元分化过程中发挥重要作用;且RA诱导后p27Kip1蛋白含量的增加是通过转录水平调节的。
OBJECTIVE: To study the differentiation of immortalized human neural progenitor cells (hSN12W-TERTcells) induced by all-trans retinoic acid (RA) and to investigate the role of cell cycle regulators p27Kip1, p21Cip1, cyclin-dependent kinase2 (cdk2) and cyclinE, to investigate the molecular mechanism of immortalized human neural progenitor cells. METHODS: The immortalized human neural progenitor cell line hSN12W-TERT cells (passage 12), established by our group, were cultured and induced with 1 μmol / L RA. The morphological changes of cells were observed on the 3rd and 7th day after RA treatment. The expressions of p27Kip1, p21Cip1, cdk2 and cyclinE mRNA in hSN12W-TERT cells before and after RA treatment were detected by RT-PCR. The expression of p27Kip1 protein was detected by immunocytochemical staining The change. RESULTS: On the 3rd day after RA induction, the hSN12W-TERT cells grew slowly and changed morphologically compared with the normal hSN12W-TERT cells without RA, which showed that the cell bodies became smaller and the number of protuberances increased. The morphological changes of hSN12W-TERT cells were more obvious at the 7th day after RA induction, close to the morphology of mature neurons. The results of RT-PCR showed that the expression of p27Kip1mRNA in hSN12W-TERT cells increased significantly after RA induction, while the expression of p21Cip1mRNA decreased slightly after RA induction. The mRNA levels of cdk2 and cyclinE in hSN12W-TERT cells did not change significantly before and after RA induction. The results of immunocytochemistry showed that the expression of p27Kip1 in hSN12W-TERT cells was significantly increased than that in normal hSN12W-TERT cells without RA (P <0.05) on the 3rd day after RA induction, and the expression of p27Kip1 was further increased on the 7th day after RA induction (P <0.05). CONCLUSION: p27Kip1 is involved in the growth arrest and differentiation of immortalized human neural progenitor cells induced by RA. P27Kip1 may play an important role in the differentiation of immortalized human neural progenitor cells; and the increase of p27Kip1 protein after RA induction Is regulated by transcriptional level.