全反式维甲酸对鼠胚神经干细胞增殖和分化的作用

THE ROLE OF ALL-TRANS-RETINOIC ACID ON THE PROLIFERATION AND DIFFERENTIATION OF RAT EM-BRYONIC NEURAL STEM CELLS

目的观察不同浓度的全反式维甲酸(all-trans-retinoic acid,ATRA)在鼠胚神经干细胞(neural stemcells,NSCs)增殖和分化中的作用,寻找促NSCs分化为神经元的较佳ATRA浓度。方法分离孕12～16d(平均15d)胚胎大鼠脑皮层,用无血清但含神经生长因子(20ng/mLbFGF、20ng/mLEGF)的培养液悬浮培养,细胞接种密度为1×106/mL,7d传代。取第3代NSCs,分别在含0.5、1.0、5.0和10.0μmol/LATRA(实验组,分别为A、B、C、D组)及不含ATRA(对照组,E组)的DMEM/F12完全培养基中培养,于培养第1、2、3、4、5、6及7天倒置相差显微镜下计数形成的细胞球数;于培养后1、3、5、7及9d采用BrdU免疫荧光染色,分析各组细胞增殖效应。另取第3代NSCs,5%FBS培养基调整细胞密度为1×106/mL,接种至6孔板内,分别加入0.5、1.0、5.0和10.0μmol/LATRA,作为各实验组(即A、B、C、D组),对照组不含ATRA(E组),于培养后3、5及7d采用免疫荧光双标染色和流式细胞仪检测各组细胞分化为神经元的比例。结果培养后1、2、3、4、5、6及7d,各实验组增殖细胞球数目接近(P>0.05),但均明显少于对照组,且差异有统计学意义(P<0.05);各实验组在培养后1、3、5、7、9dBrdU阳性细胞球增多,组间差异无统计学意义(P>0.05);对照组各时间点BrdU阳性细胞球明显多于实验组,差异有统计学意义(P<0.05)。各实验组ATRA促使NSCs分化为神经元的比例明显增高,NSE阳性细胞分化率各组间差异有统计学意义(P<0.05),其中B组最高,在培养3、5、7d分别为29.46%±0.47%、47.25%±0.46%、66.81%±0.57%,而E组NSCs主要分化为星形胶质细胞,NSE阳性细胞分化率在培养3、5、7d分别为11.11%±0.59%、14.10%±0.32%、15.92%±0.70%。流式细胞仪检测显示,培养后3、7d,促神经元分化比例各实验组与对照组比较差异均有统计学意义(P<0.05)。结论?ATRA具有显著的促NSCs分化为神经元的作用,1.0μmol/LATRA诱导分化效果最佳。

Objective To investigate the effectiveness of all-trans-retinoic acid （ATRA） at different concentrations on proliferation and differentiation of the rat embryonic neural stem cells （NSCs）, and to find the optimal concentration of ATRA that promoting the differentiation of NSCs into neurons. Methods NSCs were isolated from cerebral cortex of rat embryos （embryonic day 12-16, average 15 days）, and were cultured in serum-free medium （DMEM/F12 medium containing 20 ng/mL bFGF and 20 ng/mL EGF） at the concentration of 1× 10^6 cells/mL. Subcultures were performed 7 days after the primary culture. The cell clusters of the 3rd passage were centrifuged and divided into 5 groups. In the experimental groups （groups A, B, C, D）, the ATRA concentration was 0.5, 1.0, 5.0, 10.0 μmol/L in DMEM/F12 complete medium respectively, while in control group （group E）, the ATRA concentration was 0 in DMEM/F12 complete medium. The proliferation rate of each group was analyzed by cell counting day by day till 7th day, and BrdU positive cell counting 1, 3, 5, 7, 9 days after culture. In addition, collecting the 3rd passage NSCs and divided into 5 groups. In the experimental groups （groups A, B, C, D）, the ATRA concentration was 0.5, 1.0, 5.0, 10.0μmol/L in DMEM/F12 medium containing 5% FBS respectively, while in control group （group E）, the ATRA concentration was 0 in DMEM/F12 medium containing 5% FBS. The capacity of NSCs differentiation toward neurons was determined by immunofluorescence double-labelling and flow cytometry. Results Cell counting 1-7 days after culture in each experimental group （groups A, B, C, D） showed no significant differences （P 〉 0.05）. Cell counting at each time point of all the experimental groups were less than those of control group （P 〈 0.05）. BrdU positive cells were increased 1, 3, 5, 7, 9 days after culture in each experimental group （groups A, B, C, D）, but there was no significant difference between each experimental group （P 〉 0.05）. BrdU positive cells at each time point of control groups were more than those of all the experimental groups （P 〈0.05）. The differentiation ratio of neurons was enhanced in experimental groups and the optimal ATRA treatment concentration was 1.0 μmol/L （experimental group B）. The differentiation ratio of neurons induced by ATRA in group B was 29.460/0 ± 0.47%, 47.25% ± 0.46% and 66.81% ± 0.57% respectively after cultured 3, 5 and 7 days, whereas the differentiation ratio of neurons was 11.11% ± 0.59%, 14.10% ± 0.32% and 15.92% ± 0.70% respectively in control group. The majority of NSCs differentiated into astroglial phenotypes in control group. By flow cytometry detection, the differentiation ratio of neurons after cultured 3 days and 7 days in experimental groups were more than those in control group （P 〈 0.05）. Conclusion ATRA treatment remarkably promoted the differentiation of NSCs into neurons and the optimal concentration was 1.0μmol/L.