胎鼠海马神经元原代培养及转染条件的优化

Primary culture and the transfection conditions optimization of hippocampal neurons in fetal rat

目的建立一种稳定的大鼠胎鼠海马神经元培养方法并能够有效的鉴定其纯度,探讨用化学转染试剂体外转染大鼠胎鼠海马神经元的最佳转染条件。方法分离Wista大鼠E18d胚胎并取海马组织,无血清培养基培养并做MAP2荧光染色鉴定其纯度。应用lipofectamineTM2000转染试剂盒,大鼠神经元电转试剂盒,lipofectamineTM LTX转染试剂盒,三种不同方法包被质粒转染体外培养海马神经元。转染后观察绿色荧光蛋白的表达情况,并用台盼兰检测细胞的存活率。结果用无血清培养方法培养的海马神经元成活率好,MAP2荧光染色鉴定其纯度高于80%。三种转染方法的转染率lipofectamineTM 2000转染试剂为(14.05±2.32)%,大鼠神经元电转试剂为(52.39±1.68)%,lipofectamineLTX转染试剂为(22.87±5.32)%,其中电转试剂转染这种方法转染率最高。结论电转试剂能有效地转染体外培养的大鼠胎鼠海马神经元,同时保持很好的神经元存活率,是一种比较合适的神经元转染方法。

Objective To select a suitable primary culturing method for hippocampus neurons of newborn Wista rats and to discuss the best condition of chemical transfection agents for transfection on hippocampus neuron. Methods The hippocampus of embryo 18 d Wista rats was dissected out and the neurons were cultured and transfected by lipofectamineTM 2000, electric transfection reagents and lipofectamine TM LTX respectively. Expression of green fluorescent protein was observed and the survival rate of hippocampus neurons was determined by trypan blue after transfection. Results Serum-free culture method has a better survival rate and higher purity by MAP2 fluorescent staining. The transfection rates were （14.05±2.32）% for lipofectamineTM 2000, （52.39±1.68） % for electric transfection reagents,（22.87±5.32） % for lipofectamineTM LTX respectively. Conclusion Electric transfection is a most effective transfection method and has higher transfected rates and survival rates for cultured hippocampus neurons.