Hes1 mRNA在神经干细胞向神经细胞分化过程中表达变化的研究

Study on expression of Hes1 mRNA during the differentiation of neural stem cells toward neurons

目的探讨Hes1mRNA在神经干细胞(NSC)向神经细胞分化过程中的表达变化。方法构建乳鼠海马NSC的细胞培养模型,对NSC增殖分化进行形态学观察。细胞分化前后,采用免疫化学染色法分别检测巢蛋白(Nestin)和神经元特异性烯醇化酶(NSE)的表达,以鉴定细胞类型。流式细胞仪检测细胞周期以确定细胞增殖活性。用逆转录-PCR(RT-PCR)检测NSC中Hes1mRNA的表达变化情况。结果从海马分离培养的细胞生长状态良好,有克隆增殖能力,呈Nestin表达阳性,细胞分化后呈NSE表达阳性。流式细胞仪检测结果显示,诱导前NSC增殖活跃,其S期的细胞比例明显高于诱导分化各阶段的细胞比例(P<0.01)。与诱导前NSC相比,分化细胞各阶段G0G1期细胞比例则显著增多(P<0.01),细胞停滞于G0G1期。RT-PCR结果表明,Hes1mRNA在NSC诱导分化前后均有表达,与诱导前相比,分化后的各阶段Hes1mRNA表达量均明显减少(P<0.05),而分化各阶段Hes1mRNA表达量差异无统计学意义(P>0.05)。结论 Hes1mRNA的高表达可能与NSC的增殖有关,而Hes1mRNA表达的减弱则有利于神经细胞的大量分化。

Objective To explore the expression of Hes1 mRNA during neural stem cells（NSC） differentiation toward neurons . Methods To establish the model of cultivation NSC in the hippocampal of newborn （24 h） SD rats ,and then to observe the mor-phology of NSC in the course of proliferation and differentiation .Before and after cellular induction ,the expression of Nestin and NSE were respectively measured to detect cell types by immunochemistry method .And flow cytometry was used to determine cell cycle phases ,so as to detect proliferative activity of these cells .Meanwhile ,the expression of Hes1 mRNA in NSC was determined by reverse transcription-PCR（RT-PCR） .Results The results demonstrated that NSC isolated from hippocampal showed vigorously clonal proliferation in vitro ,and positive Nestin expression .In addition ,the differentiated cells demonstrated positive NSE expres-sion .Flow cytometry analysis showed that the percentage of NSC in S phase was obviously higher than that of induced differentia-tion of all time（P〈0 .01） ,which indicated that NSC were actively dividing induction before .Compared to NSC ,the percentages of cells in G0 G1 phases were increased significantly after neuronal differentiation （P〈0 .01） ,which indicated that differented cells have arrested in G0 G1 phases .Meanwhile ,the results from RT-PCR showed that ：Hes1 mRNA was expressed in NSC both before and af-ter induced differention .Compared to induction before ,the level of Hes1 mRNA expression in NSC during different stages of differ-entiation after induction were significantly decreased （P〈0 .05） ,and Hes1 mRNA did not show any obvious changes among these stages of differentiation（P〉0 .05） .Conclusion The high level of Hes1 mRNA was probably involved proliferation of NSC .How-ever ,low level of Hes1 mRNA might contribute to neuronal differentiation .