大鼠肠肌间神经丛神经细胞原代培养方法的建立

Establishment of primary culture method for intestinal myenteric plexus neurons in rats

目的建立一种操作简单、稳定性好的大鼠肠肌间神经丛神经细胞的原代培养方案。方法2~4周龄健康雄性SD大鼠,取小肠分离出纵行肌间神经丛,使用Ⅱ型胶原酶和胰蛋白酶消化,获取大鼠肠肌间神经丛神经细胞,使用含神经生长因子(GDNF)的Neurabasal A培养基进行培养。进行细胞形态学观察及细胞免疫荧光染色鉴定,膜片钳实验检测肠神经元细胞的静息膜电位和膜电阻。结果原代培养的大鼠肠神经元细胞生长良好。培养5~7 d的肠神经元细胞可见长的突起,神经元和神经胶质细胞网络紧密排列;培养45 d显示细胞皱缩、神经突触断裂,呈细胞凋亡表型。取培养5 d的肠神经元细胞进行膜片钳实验,记录其静息膜电位为(-49.5±1.5)mV、膜电阻为(500±38)MΩ(n=30)。结论本原代培养方案取材容易、培养方便、简单易行,细胞接种培养5~7 d可用于单细胞膜片钳实验,为深入研究神经细胞提供了可靠模型。

Objective To establish an easy-to-use,stable method for primary culture of intestinal myenteric plexus neurons in rats.Methods Longitudinal myenteric plexus was harvested from 2-4 week old male Sprague Dawley(SD)rats,and then digested with collagenaseⅡand trypsin to obtain intestinal myenteric plexus neurons.The neurons were cultured in Neurabasal A medium supplemented with glial derived neurotrophic factor(GDNF).The neurons were examined by morphological observation,and validated with immunofluorescent staining.The resting membrane potential and resistance of the neurons were measured by patch clamping.Results The myenteric plexus neurons of SD rats good growth.At 5-7 days of cuture,long processes were seen to sprout from the neurons which were closely aligned with glial cells in a network.At 45 days of culture,the neurons showed apoptotic phenotype as reflected by cell shrinkage and synaptic fragmentation.Patch clamping with myenteric plexus neurons at 5 days of culture showed a resting membrane potential of(-49.5±1.5)mV and a membrane resistance of(500±38)MΩ(n=30).Conclusion This method of primary culture is well accessible to sample procurement,facilitative for culture,and easy to use.Inoculated cells cultured for 5-7 days are suitable for use in single-cell patch clamping,which provides a reliable model for further study of neurons.