小鼠神经母细胞瘤N2a细胞不同分化培养方法的比较

Comparison of different methods for differentiation of murine neuroblastoma N2a cells

目的:比较5种不同分化培养方法对小鼠神经母细胞瘤N2a细胞形态、分化情况、分化标志物及增殖、凋亡的影响。方法:N2a细胞分别以D10[DMEM+10%胎牛血清(FBS)]完全培养基和不同分化培养基D1(DMEM+1%FBS)、D0(不含FBS的DMEM培养基)、30%OM(DMEM+30%Opti-MEMⅠ)、D2+视黄酸(RA)(DMEM+2%FBS+10μmol·L-1 RA)和D1+RA(DMEM+1%FBS+10μmol·L-1RA)培养后显微镜眀场随机拍照,观察不同时间点的分化比例、突起长度及突起数量;免疫荧光染色和蛋白质印迹法检测神经元标志物的表达;EdU掺入流式细胞术、CCK-8和AnnexinⅤ/PI双染流式细胞术检测不同培养方法对细胞增殖和凋亡的影响。结果:中等密度培养条件下D0组72 h分化比例为(35.92±2.63)%,D1、30%OM、D2+RA和D1+RA组96 h分化比例分别为(10.40±2.05)%、(29.70±4.98)%、(11.37±2.56)%和(45.13±8.75)%;分化后神经元标志物微管蛋白beta-Ⅲ(βⅢ-tubulin)和生长相关蛋白43(GAP43)表达上调。D0组分化培养24 h后即可见长突起,突起以单支为主,但增殖能力较D10和30%OM组低,凋亡和死亡比例高,72 h后细胞状态差,大部分死亡。D1+RA组分化培养24 h也可见长突起,细胞呈多支生长,增殖能力也较D10和30%OM组低,凋亡和死亡比例高。30%OM组分化72 h才见较长突起,突起以两支为主,细胞增殖能力较D10组略低,但凋亡与D10组无显著差异。在低密度培养条件下30%OM可实现长时间分化培养,分化比例随培养时间的延长而增加,培养144 h分化比例达(56.22±6.27)%,突起长度(176.73±108.61)μm。结论:D0、30%OM和D1+RA分化培养方法均可实现较高比例的分化,D0和D1+RA分化和长突起的出现早于30%OM,但细胞增殖率低,凋亡比例高,不利于长时间培养;30%OM培养在低密度下可实现高分化比例和长突起,适合长时间分化培养。

Objective: To compare the morphology, differentiation, markers, proliferation and apoptosis of murine neuroblastoma N2 a cells induced with five different differential culture medium. Methods: N2 a cell line was grown in complete medium [DMEM+10% fetal bovine serum(FBS)]and was replaced with five different differential medium: D1(DMEM+1%FBS), D0(DMEM without FBS), 30%OM(DMEM+30%Opti-MEMⅠ), D2+retinoic acid(RA)(DMEM+2%FBS+10 μmol·L-1RA) and D1+RA(DMEM+1%FBS+10 μmol·L-1RA) to allow for differentiation. The differentiation ratio, the length and numbers of neurites at different time points were measured by photographs shown in bright field, the expression of neuron markers were detected by immunostaining and Western blotting, the effects of different differential medium on cell proliferation and apoptosis were detected by EdU labeling, CCK-8 and Annexin Ⅴ/PI double staining assays. Results: The differentiation ratio of N2 a cell line induced with D0 at the time of 72 h was(35.92±2.63)%, D1, 30%OM, D2+RA and D1+RA under medium density at 96 h were(10.40±2.05)%,(29.70±4.98)%,(11.37±2.56)% and(45.13±8.75)% respectively, and the expression of neuron marker βⅢ-tubulin and grouth associated protein 43(GAP43) were up regulated in differentiated N2 a cells. N2 a differentiation induced with D0 had long single neurite at the time of 24 h, but the ability of proliferation was much lower than that of D10 and 30%OM, and the proportion of apoptosis and death were much higher, after 72 h of inducing, N2 a cells were poor and most of them died. N2 a differentiation induced with D1+RA showed long but multiple neurites, lower proliferative capacity and more apoptotic cells. N2 a differentiation induced with 30%OM showed long neurites later than D0 and D1+RA, and most of the differentiated cells had two neurites. Unlike D0 and D1+RA, 30%OM showed slightly lower proliferation capacity and no significant difference of apoptosis than complete medium D10. Moreover, N2 a cells could be differentiated by 30%OM for longer than 144 h under low density, the differentiation ratio and the length of the neurites reached to(56.22±6.27)% and(176.73±108.61) μm respectively. Conclusion: D0, 30%OM and D1+RA differential medium can all induce N2 a differentiation to a higher ratio, D0 and D1+RA show earlier long neurite outgrowth, but lower proliferation capacity and higher apoptotic ratio than 30%OM, which is not conducive to long time culture. The differentiation induced by 30%OM can also achieve to a high differentiation ratio and long neurite under low density, moreover, it’s suitable for long term culture.