摘要
目的介绍改良的Banker共培养方法,并通过膜片钳、原子力显微镜检验神经元的生长状态。方法利用烙铁在培养皿底面滴入4个高度约为0.1mm的石蜡,以支持盖玻片建立共培养体系。当星形胶质细胞生长至底面积70%~80%后,将胰酶消化、分离的神经元接种在盖玻片上,4h后翻转盖玻片并移到含有星形胶质细胞的培养皿内,进行共培养,4~5d半量换液。膜片钳记录神经元动作电位和自发性突触后电位,原子力显微镜观察培养25d的神经元生长状态。结果神经元最长培养40d,接种后4h^6d和9~13d是两个快速增长期。膜片钳实验中,神经元能够产生动作电位,并且随刺激强度增大动作电位频率增加;能够记录到神经元的兴奋性突触后电位(EPSP)、抑制性突触后电位(IPSP)和自发性动作电位(sAP)。原子力显微镜观察可见神经元胞体呈典型锥体形状,细胞表面光滑平整。结论神经元和胶质细胞共培养方法稳定性强,容易操作,培养的神经元存活时间长、生长状态良好。
Objective To introduce a modified Banker＇s co-cuhure method used for neurons culture. Methods In order to support the coverslip, four drops of paraffin about 0. 1 nun high were made by soldering ion in the bottom of dish. When astrocytes covered 70% -80% of the dish bottom, the dissociated neurons by trypsin were cultured on the coverslip. Four hours later, the eoverslip was inverted and transferred to the dish containing astrocytes, and the half of mediunv was exchanged every 4 to 5 d. The action potential （AP） and spontaneous postsynaptic potential （sPSP） were recorded by patch clamp and the neurons were observed by atomic force microscopy （AFM）. Results The neurons survived for 40 d, the longest time of culture had two rapid development period on 4 h to 6 d and 9 to 13 d. In patch experiment, AP could be recorded and the frequency of AP was enhanced with the increase of stimulation intensity. The typical sPSP including excitatory postsynaptic potential （EPSP）, inhibitory postsynaptic potential （IPSP） and spontaneous action potential （SAP） also could be recorded. The data of AFM indieated that the membrane surfaces of neurons were smooth. Conclusion The method of co-cuhure is stable and easy to master, and the cultured neurons can survive long time in good state.
出处
《中华神经外科疾病研究杂志》
CAS
2006年第4期317-320,共4页
Chinese Journal of Neurosurgical Disease Research
关键词
共培养
膜片钳
原子力显微镜
Co-culture
Patch clamp
Atomic foree microscopy
