摘要
为了进一步了解原代培养神经元技术是否可以用于建立可靠的乙酰胆碱能神经元体外培养细胞模型,该实验检测了分别培养自E18胎鼠基底前脑(basal forebrain, BF)和海马(hippocampus,HIP)原代培养神经元中的乙酰胆碱能神经元的标志物,同时比较了离体培养细胞与在体在相同脑区中乙酰胆碱能标志物表达的差异。使用免疫标记荧光方法,检测了来自E18胎鼠的基底前脑脑区和海马脑区的离体原代培养神经元中在DIV 3和DIV 21时间点上表达Ch AT和p75NTR(两种常用的胆碱能神经元标记物)的神经元的数量,分析其占总神经元数量的比例,并与E18胎鼠和成年小鼠相同脑区的在体组织切片中的结果相比较。结果显示, Ch AT和p75NTR均在来自基底前脑和海马的培养神经元的DIV 3和DIV 21中高比例表达。然而,虽然在E18胎鼠和成年小鼠的基底前脑的组织切片中有Ch AT和p75NTR的表达,但是在同时期的海马组织切片中并无Ch AT的表达,并且来自基底前脑和海马脑区的培养神经元中表达乙酰胆碱能神经元标志物的神经元数量占总神经元数量比例与在体并不一致。这些结果显示,乙酰胆碱能标志物在离体原代培养和在体中的表达状况可能存在不同。根据实验结果推测,在体外应用原代培养方法培养乙酰胆碱能标志物免疫阳性神经元可能并不是乙酰胆碱能神经元。除了通过免疫组织化学方法,还需要更多的技术和方法来鉴定培养细胞中的乙酰胆碱能神经元。
To further understand whether primary cultured cholinergic neurons can be used as a viable cell model for cholinergic neurons in vitro, we compared differences in proportion of neurons expressed cholinergic biomarkers and total neurons in cultured neurons cultivated from E18 basal forebrain(BF) and E18 hippocampus(HIP), and the differences in expression ratio of cholinergic markers between primary culture neurons and brain slices in the same brain regions. Immunofluorescence was used to detect the proportion of Ch AT and p75 NTR(two commonly used cholinergic neuron markers) expression in DIV 3 and DIV 21 of cultured neurons of basal forebrain and hippocampus from E18 fetus, and also in E18 fetus and adult mice in brain slice. Both Ch AT and p75 NTR showed high expression ratio in cultured neurons from BF and HIP in DIV 3 and DIV 21. However, there was no expression of Ch AT in hippocampal slices of E18 fetus and adult mice. Therefore, the ratio of neurons expressed cholinergic markers in cultured neurons from basal forebrain and hippocampus is inconsistent with in vivo, suggesting that it is temporarily not feasible to culture primary cholinergic neurons in vitro by using primary neuronal culture methods. Meanwhile, in addition to immunostaining more techniques and methods are needed to identify cholinergic neurons in cultured cells.
作者
李一梅
王国祥
王云
Li Yimei;Wang Guoxiang;Wang Yun(Institutes of Brain Science Fudan University,Shanghai 200032,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2019年第8期1543-1550,共8页
Chinese Journal of Cell Biology
基金
supported by the National Natural Science Foundation of China(Grant No.31471027)~~
