体外原代培养小鼠胚胎背根神经节细胞的神经化学特征

Neurochemical characteristics of cultured primary neurons from embryonic mouse dorsal root ganglia

目的探讨胚胎背根神经节(DRG)感觉神经元作为体外研究神经多肽的细胞模型之有效性。方法采用免疫荧光及相差显微技术对30只胚胎小鼠背跟神经节细胞选择性神经多肽的分布、细胞大小及其多肽表达与细胞大小之间的关系进行观察比较。结果培养时间达3周龄的DRG细胞主要以中小直径(30～20μm)的细胞为主,与成年在体脊髓DRG细胞的形态多形性特征类似;选择性神经多肽(钙调素基因相关多肽、P物质、甘丙肽和nociceptin)的表达也随着体外培养时间的延长明显增强,且从早期仅在胞体部位表达到3周时细胞周围神经突起也出现显著免疫荧光阳性。此外,体外培养达到3周时,降钙素基因相关多肽和P物质主要在体积较小的神经细胞表达,与成年鼠DRG的分布特征一致。而甘丙肽与nociceptin在不同大小DRG神经元的表达没有像降钙素基因相关多肽和P物质一样随着培养时间的延长而出现明显改变。结论胚胎小鼠DRG神经细胞培养可以作为研究感觉神经细胞某些重要的神经多肽(降钙素基因相关多肽,P物质)在调节感觉神经细胞内相关信号转导通路中作用的体外模型。

Objective Morphological heterogeneity and the expression of neuropeptides in the cuhured primary neurons of the dorsal root ganglia from the embryonic mouse were investigated. Methods Morphology and neurochemistry of cultured dorsal root ganglia （DRG）neurons were analyzed by phase-contrast microscopy and immnuofluorensence. Results Morphological characteristics of DRG neurons with a 3-week culture period appeared similar to those observed in adult DRG neurons. The ratio of median to small sized neurons （20 -30μm） was increased from （8.9 ± 0. 61 ）% after 1- week culture to （29.58-± 1.23）% after 3-weeks culture. The selective neuropeptides, CGRP, substance-P, galanin and nociceptin, were expressed in neuron soma from the first week of the culture. After 3 weeks of culture, an obvious immunoflurensent express of the peptides in neuronal processes was observed. In addition, most neurons containing calicitonin gene related peptide（CGRP） and substance-P belonged to small-size DRG neurons in 3-weck-old DRG culture, which was similar to the features of adult spinal DRG neurons. The morphological features of galanin- and noeiceptin- positive neurons didn＇ t show any difference from 1-week to 3-week culture. Conclusion The embryonic mouse DRG neuron culture might be set up as an in vitro model for the study on some important sensory neuropeptides, like CGRP, substance-P, and their roles in modulating intracellular signaling pathways for sensation.