一种简单的原代神经元培养及转染方法

Culture and lentiviral transfection of primary neurons in vitro

目的建立一种简单、高效、稳定的原代神经元培养及转染方法。方法无菌条件下分离生后2 d内SD大鼠海马,经消化、反复沉淀后获得海马组织细胞悬液,利用差速贴壁法对神经元进行纯化,采用神经元专用无血清培基进行培养,并于倒置显微镜下观察神经元形态的变化,采用微管相关蛋白（MAP2）抗体对细胞进行鉴定;利用慢病毒转染神经元,观察神经元转染效率。结果体外培养的原代神经元,在培养第10~15天可以形成密集的神经元网络,细胞健壮,神经元特征明显;经MAP2鉴定,神经元纯度达95%以上;利用慢病毒转染神经元,转染效率达95%以上。结论采用该方法进行原代神经元培养,能够获得高纯度的原代神经元;慢病毒是神经元转染的理想载体。

Objective To establish a simple,efficient and stable method of primary neurons＇ culture and transfection. Methods The hippocampus of SD rats within 2 days after birth was separated under aseptic condition, which was digested and received repeated deposition to gain the hippocampus cell suspension. The neurons were purified by differential adhension method and were cultured with serum-free medium. The morphological changes of neurons were observed by inverted microscope. Microtubule-associated protein （MAP2） antibodies were used to identify the ceils. The lentiviral transfected neurons were used to observe the efficiency of neuron transfection. Results The dense networks of primary neurons,which were cultured in vitro,were formed 10 to 15 days after culture, with healthy cells and specific characteristics of the neurons. The purity of neurons was more than 95% identified by MAP2. The transfection efficiency achieved more than 95% using lentiviral transfected neurons. Conclusion The method we have used to culture primary neurons can be adopted to obtain highly purified neurons. The lentiviral is an excellent carrier for neuron transfection.