【摘要】 目的观察全反式视黄酸(RA)对体外培养的人神经前体细胞的诱导作用,研究在这个过程中细胞周期调节蛋白p27Kip1、p21Cip1及相关分子cdk2、cyclinE的变化,探讨p27Kip1在RA诱导的人神经前体细胞分化过程中的作用。方法取12wk的人胚胎前脑纹状体,原代培养人神经前体细胞。在细胞进入对数生长期时给予1μmol·L-1RA诱导。在诱导的d1、3、7,用相差显微镜观察细胞形态的变化;用免疫细胞化学染色、RT-PCR等方法检测p27Kip1、p21Cip1及其相关的cdk2、cyclinE的变化。结果在RA诱导d3,人神经前体细胞形态发生变化,至d7时已接近成熟神经元形态。免疫细胞化学染色结果显示p27Kip1的表达在RA诱导d3增加,在RA诱导d7时明显增加,与未经RA诱导的细胞相比差异有统计学意义(P<0.05)。RT-PCR结果显示,p27Kip1 mRNA的表达在RA诱导d3增加,并在RA诱导d5达到高峰,而p21Cip1 mRNA的表达在RA诱导后略呈下降趋势。cdk2、cy-clinE的mRNA水平在RA诱导前后没有明显变化。结论p27Kip1在RA诱导的人神经前体细胞分化过程中具有重要作用,并且其表达增加是通过转录水平调节的。更多还原

【Abstract】 Aim To investigate whether human neural progenitor cells（hNPCs）can be induced to differentiate toward a neuronal phenotype by all-trans Retinoic acid（RA）,and whether there is any functional link between p27^Kip1function and RA in the control of neuronal differentiation in hNPCs.Methods hNPCs 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 appropriate time,hNPCs were exposed to 1 μmol·L-1 RA for 1,3,5,7days respectively.The properties of hNPCs were characterized by using phase-contrast microscopy、immunocytochemistry and RT-PCR.Results To monitor morphological changes induced by RA,RA-treated hNPCs were checked by phase-contrast microscopy.After 3 days treatment with RA,the cells began to show phenotypical changes,assuming a neuronal-like morphology with contemporaneous formation of dendritic-like structures.Mature neuronal-like morphology was observed after being exposed to RA for 7 days.By immunocytochemistry,the expression of p27^Kip1was elevated after exposure to RA for 3 days compared with that of normal untreated hNPCs,and significantly increased after exposure to RA for 7 days（P<0.05）.By RT-PCR,p27^Kip1mRNA was elevated after exposure to RA for 3 days,with a peak at 5 days.The expression of p21Cip1 mRNA was weak and decreased very little following RA treatment.No difference was found for the expression of cdk2 and cyclinE mRNA before and after RA treatment.Conclusions There is a functional link between RA and p27^Kip1function in the control of neuronal differentiation in hNPCs.p27^Kip1plays a key role during neuronal differentiation.Moreover,high levels of p27^Kip1are regulated via increased p27^Kip1mRNA expression.更多还原