原代海马神经细胞体外培养的纯化与鉴定

Purification and identification of hippocampal neurons primary cultured in vitro

目的建立原代海马神经细胞体外培养纯化及鉴定的优选方法。方法取新生Wistar乳鼠,分离海马后在体外采用含血清结合无血清法进行培养,并用NSE、GAP-43、MAP2等海马神经细胞特异抗体经免疫细胞化学方法鉴定细胞性质及纯度。结果接种24 h后细胞全部贴壁,并长出突起,随时间增加,突起延长并交错形成网络,至第7-8天神经元形态最为成熟饱满,随后逐渐出现细胞老化,神经元最长可生存4周。经鉴定,海马神经元纯度达96%以上。结论体外采用含血清和无血清法相结合进行原代海马神经细胞培养,细胞纯度高,杂细胞少,可为神经疾病体外研究提供必要细胞基础。

Objective To establish an optimizing pal neurons primary cultured in vitro. Methods method of purification and identification of hippocam- Hippocampus was disconnected from new-born rat and then hippocamal neurons were cultured with both serum medium and serum-free medium in vitro. Identification and purification of neurons was detected with specific NSE,GAP-43 and MAP2 antibodies by immunocytochemistry. Results All cells adhered to the footwall after 24 hours and short nervous processes were found in subtotal neurons, and these processes extended markedly with time and connected into nets. Cells were fully developed on the 7th -8th day, and then began aging; 4 weeks were the longest life span observed. Hippocampal neurons got more than 96% purity after identifying. Conclusion Primary culture of hippocampal neurons with serum medium and serum-free medium in vitro is with high purity and low heterogeneity, as can be used as cell basis in research of nervous diseases in vitro.