一种成年SD大鼠背根节神经元分离与培养新方法的建立

Dissection and Culture of Dorsal Root Ganglion Neurons from Adult SD Rats

目的:建立成年SD大鼠背根神经节神经元的分离和培养的方法,并探索胶原酶的最佳消化时间。方法:取成年Sprague-Dawley(SD)大鼠的背根神经节(Dorsal Root Ganglion,DRG),经0.5%的胶原酶Ⅰ和0.05%胰酶消化后,接种到多聚赖氨酸包被的细胞培养板中;4 d后传代并用差速贴壁法纯化神经元细胞;采用神经元特异性烯醇化酶(Neuron-specific Enolase,NSE)免疫组化试剂盒进行鉴定,并计算细胞纯度;分别用胶原酶作用1h、24 h和48 h来探索胶原酶消化的最佳时间。结果:培养的DRGs生长状态良好,传代一次后杂细胞多数被去除,神经元的纯度可达到(94.28±1.87)%,贴壁的DRG神经元可存活2至3周。结论:成功建立成年SD大鼠DRG神经元原代培养体系,该体系可获得生长状态良好、纯度较高的DRG神经元细胞。

Objective： To establish a dissection method and a purification culture system of dorsal root ganglion（ DRG）neurons from adult Sprague- Dawley（ SD） rats and to explore the best digestion time of collagenase to the DRGs. Methods： DRGs obtained from adult SD rats were digested by 0. 5% collagenase I and 0. 05% trypsin,and then inoculated into cell culture plates coated by poly- L- Lysine. The DRG neurons were sub- cultured 4 days later and purified by differential adhesion. The neuronal- specific enolase（ NSE） immunohistochemical kit was used to identify the DRG neurons and the purity coefficient was evaluated according to results of cell count. To explore the best digestion time of collagenase,the equal amount of DRGs were digested by collagenase for 1 h,24 h and 48 h respectively. Results： The cultured DRG neurons from adult SD rats grew healthily and survived 2 to 3 weeks. The purity coefficient of DRG neurons was（ 94. 28 ± 1. 87） %. Conclusion： We established the primary culture system of DRG neurons of adult SD rats successfully and by which we can get the DRG neurons with high purity in good condition.