Yun Hyeok Jun, Kim Eun-Hye, Kim Byung Gon
Department of Brain Science, Ajou University School of Medicine; Neuroscience Graduate Program, Department of Biomedical Sciences, Ajou University School of Medicine.
Department of Brain Science, Ajou University School of Medicine; Neuroscience Graduate Program, Department of Biomedical Sciences, Ajou University School of Medicine; Department of Neurology, Ajou University School of Medicine;
J Vis Exp. 2018 Mar 30(133):56920. doi: 10.3791/56920.
There is strong evidence that macrophages can participate in the regeneration or repair of injured nervous system. Here, we describe a protocol in which macrophages are induced to produce conditioned medium (CM) that promotes neurite outgrowth. Adult dorsal root ganglion (DRG) neurons are acutely dissociated and plated. After the neurons are stably attached, peritoneal macrophages are co-cultured on a cell culture insert overlaid on the same well. Dibutyryl cyclic AMP (db-cAMP) is applied to the co-cultures for 24 h, after which the cell culture insert containing the macrophages is moved to another well to collect CM for 72 h. The CM from the co-cultures treated with db-cAMP, when applied to a separate adult DRG neuron culture, exhibits robust neurite outgrowth activity. The CM obtained from the db-cAMP-treated cultures consisting of single cell type alone, either DRG neuron or peritoneal macrophage, did not exhibit neurite outgrowth activity. This indicates that the interaction between neurons and macrophages is indispensable for the activation of macrophages secreting molecular factors with neurite outgrowth activity into CM. Thus, our co-culture paradigm will also be useful to study intercellular signaling in the neuron-macrophage interaction to stimulate the macrophages to be endowed with a pro-regenerative phenotype.
有强有力的证据表明巨噬细胞能够参与受损神经系统的再生或修复。在此，我们描述一种方案，即诱导巨噬细胞产生促进神经突生长的条件培养基（CM）。将成年背根神经节（DRG）神经元急性解离并接种。待神经元稳定附着后，将腹腔巨噬细胞与覆盖在同一孔上的细胞培养插入物共培养。将二丁酰环磷腺苷（db - cAMP）应用于共培养物24小时，之后将含有巨噬细胞的细胞培养插入物移至另一孔中收集CM 72小时。用db - cAMP处理的共培养物的CM，当应用于单独的成年DRG神经元培养物时，表现出强大的神经突生长活性。从仅由单一细胞类型（DRG神经元或腹腔巨噬细胞）组成的经db - cAMP处理的培养物中获得的CM未表现出神经突生长活性。这表明神经元与巨噬细胞之间的相互作用对于激活巨噬细胞向CM中分泌具有神经突生长活性的分子因子是不可或缺的。因此，我们的共培养模式对于研究神经元 - 巨噬细胞相互作用中的细胞间信号传导以刺激巨噬细胞获得促再生表型也将是有用的。
