摘要
目的为缺血性脑损伤模型的制备及研究建立一种更好的体外培养海马神经元的方法。方法实验组取出生24h内的Wistar新生鼠海马,用木瓜酶和DNA酶消化,以1×108/L的密度接种于含体积分数0.20胎牛血清的DMEM-F12培养液中,24h后更换无血清培养液,每隔3d半量换液。对照组采用传统的培养方法培养细胞。培养期间用倒置显微镜观察细胞形态及数量变化。采用免疫荧光法测定神经元特异性烯醇化酶表达,判断海马神经元培养是否成功。结果实验组的海马神经元培养12h,可见大部分细胞贴壁;培养24h,大部分细胞可见3-4个突起,突起长度为（25.0±2.5）μm;培养3d,神经元胞体增大,可见明显光晕;之后神经元分化逐渐成熟,胞体透亮,呈锥形或多极形,光晕明显,神经元之间的突起联系更加紧密,形成密集的神经细胞网络;培养13d后,神经元细胞开始退化、变性,胞体萎缩,神经细胞网络开始老化。随着培养天数的增加,神经元可出现少量凋亡,但实验组培养1、3、5、8d时的神经元数量明显高于对照组（t=11.5-49.5,P〈0.05）。免疫荧光法鉴定显示,实验组神经元细胞阳性率为（93.53±1.67）%。结论用上述改良方法培养得到的海马神经元细胞生长状态良好,可用于后续实验研究。
Objective To establish a better method for primary culture of hippocampal neurons in vitro for preparation and study of the model of ischemic brain injury（IBI）. Methods This study was carried out in two groups as experimental group and control group.In the experimental group,the hippocampus of new-born Wistar rats within 24 hours was collected and digested with papaya enzyme and DNA enzyme.The hippocampus cell suspension of a 1×108/L concentration was inonculated to DMEMF12 medium containing 0.20 fetal bovine serum and the medium was replaced into serum-free medium,and then,half of it was changed every three days.In the control group,the cells were calculated with traditional method.During the culture,the morphology of the neurons was observed using an inverted microscope.Immunocytochemical methods were used to detect the neuron-specific enolase（NSE）and to judge whether the hippocampus neurons culture was successful. Results After 12 hours of cultivation,cell attachment was mostly seen in the experimental group.After 24 hours,three or four protrusions with neurite length of（25.0±2.5）μm could be seen.After three days,the neural bodies enlarged with clear halo.The neuron differentiation was then gradually maturated and exhibited translucent cell body as conical or multipolar shape with obvious halo.The process between neurons was more closely linked and thus forming dense neural networks.After 13 days of culture,degereration of neuronal cells began,cell body atrophy,and network started aging.With the increase of days of culture,a small amount of apoptosis could be seen in neurons.After 1,3,5and 8days of culture,the number of neurons of the experimental group was higher than that of the control group（t=11.5-49.5,P〈0.05）.Immunofluorescence results showed positive cells of the experimental group accounted for（93.53±1.67）%. Conclusion The hippocampal neurons that were cultured with the above modified method grow well,and can be used for subsequent experimental study.
出处
《青岛大学医学院学报》
CAS
2016年第5期519-521,共3页
Acta Academiae Medicinae Qingdao Universitatis
基金
国家自然科学基金项目(81371448)
