p27^(Kip1)在永生化人神经前体细胞分化中的作用研究

Study on the role of p27^(Kip1) during the differentiation of immortalized human neural progenitor cells

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 changes of cell cycle regulatory proteins p27Kip1, p21Cip1, cyclin-dependent kinase2 （cdk2） and cyclinE during all-trans retinoic acid （RA）-induced differentiation of immortalized human neural progenitor cells （hSN12W-TERT cells）, in order to investigate the molecular mechanism that controls human neural progenitor cells differentiation. Methods： Immortalized human neural cell line, hSN12W-TERT, was established by our research group before. hSN12W-TERT cells （the 12th passage） were cultured and treated with 1 μmol/L RA for 3 and 7 d respectively. The morphological changes of hSN12W-TERT cells were observed following RA treatment. The expression of p27Kip1, p21Cip1, cdk2 and cyclinE mRNA in hSN12W-TERT cells before and after RA treatment cells were determined by using RT-PCR analysis. Immunocytochemical staining was used to further determine the expression of p27Kip1 before and after RA treatment. Results： Following 3-day-RA treatment, the hSN12W-TERT cells grew slowly and began to show morphological changes with smaller cell body and longer, more processes compared with normal untreated hSN12W-TERT cells. Mature neuronal-like morphology was observed after exposed to RA for 7 d. RT-PCR analysis results showed that the expression of p27Kip1 mRNA was elevated significantly following RA treatment, while the expression of p21Cip1 mRNA decreased a little bit following RA treatment. The expression of cdk2 and cyclinE mRNA in hSN12W-TERT cells before and after RA treatment was similar. Immunocytochemical staining results showed that the expression of p27Kip1 was elevated following 3-day-RA treatment compared with that of normal untreated hSN12W-TERT cells （P0.05）. The expression of p27Kip1 was further increased after exposed to RA for 7 d （P0.05）. Conclusion： p27Kip1 is associated with RA-induced growth arrest and neuronal differentiation of immortalized human neural progenitor cells. p27Kip1 might play a key role during neuronal differentiation. Moreover, high protein levels of p27Kip1 in hSN12W-TERT cells following RA treatment are probably regulated via increased p27Kip1 mRNA expression.