新生Long Evans大鼠初级视皮层神经元的培养和鉴定

Identification and culture of visual cortex neurons in newborn Long Evans rats

目的建立新生Long Evans大鼠视皮层神经元原代培养方法。方法采用钝性分离1d龄Long Evans大鼠双侧视皮层,2.5g·L-1胰蛋白酶消化法获得细胞悬液,在无血清培养基中进行培养,7～14d后行免疫组织化学及膜片钳鉴定。结果用该方法培养的视皮层神经细胞存活率高,生长状态良好,存活可达1个月左右,β-III tubulin免疫组织化学染色阳性神经元占培养细胞中的80%以上,膜片钳成功记录了180个细胞,静息膜电位在-50～-70mV,存在内向、外向电流,脉冲恒流刺激可诱发多个动作电位。结论本研究所采用的分离及原代培养方法是一种简单、高效的视皮层神经元培养方法。

Objective To establish the protocol for culturing visual cortex neurons derived from newborn Long Evans rats in vitro.Methods Tissues from visual cortex of one day aged Long Evans rats were harvested and digested into cell suspension by 2.5 g·L-1 trypsogen.Those cells were then cultured with serum free medium,the cultured cells were identified by immunochemical staining and patch clamp recording after cultured from 7 days to 14 days.Results The neurons from visual cortex had high survival rate and good conditions during growth,some cells could survive one month.The positive cells staining of β-III tubulin were more than 80%,180 cultured visual cortex neurons were successful recorded by patch clamp,the resting membrane potentials were between-50 mV and-70 mV,inward current and outward current were existed,several action potentials could be elicited by constant current stimulation.Conclusion This method is simple and effective for the culture of visual cortex neurons.