Tao-Cheng J H, Dosemeci A, Bressler J P, Brightman M W, Simpson D L
NINDS EM Facility, National Institutes of Health, Bethesda, MD 20892, USA.
J Neurosci Res. 1995 Oct 15;42(3):323-34. doi: 10.1002/jnr.490420306.
PC12 cells can differentiate into neuron-like cells after treatment with either nerve growth factor (NGF) or transduction with a retrovirus which expresses the K-ras oncogene. The concomitant treatment of NGF plus ras differentiates PC12 cells further than either agent alone with respect to neurite outgrowth, acetylcholinesterase levels, and most strikingly, the number of synaptic vesicle (SV) clusters. These SV clusters in PC12 cell neurites closely resemble those in the presynaptic terminals of neurons. Such SV clusters have not been described in cell lines previously. The SV clusters from all three differentiated groups (NGF, ras, and NGF plus ras) were similar in size, shape, and configuration, except that the ones in the doubly treated group occur in higher frequency and have more vesicles. The synaptic nature of these vesicle clusters was demonstrated by their regulated depletion after potassium stimulation. Furthermore, these vesicle clusters stained positively for two SV-associated proteins, synapsin I and synaptophysin, by EM immunocytochemistry (ICC). Such SV clusters in a cell line are very useful for characterizing the regulated release of SVs and the distribution of SV-related antigens in intact cells. Analysis by SDS-gel electrophoresis and immunoblotting indicated that synapsin I levels are higher in all three differentiated groups compared to untreated cells; whereas synaptophysin levels are lower in cells exposed to NGF alone or with NGF and ras double treatment. Possible convergence and/or divergence on the mechanisms of NGF and ras differentiation in PC12 cells are discussed.
用神经生长因子（NGF）处理或用表达K-ras癌基因的逆转录病毒转导后，PC12细胞可分化为神经元样细胞。与单独使用任何一种试剂相比，NGF加ras的联合处理在神经突生长、乙酰胆碱酯酶水平方面，最显著的是在突触小泡（SV）簇数量方面，能使PC12细胞进一步分化。PC12细胞神经突中的这些SV簇与神经元突触前终末中的SV簇非常相似。这种SV簇以前在细胞系中尚未有过描述。来自所有三个分化组（NGF、ras和NGF加ras）的SV簇在大小、形状和结构上相似，只是双重处理组中的SV簇出现频率更高且有更多小泡。钾刺激后这些小泡簇的调节性耗竭证明了它们的突触性质。此外，通过电子显微镜免疫细胞化学（ICC），这些小泡簇对两种与SV相关的蛋白——突触素I和突触囊泡蛋白呈阳性染色。细胞系中的这种SV簇对于表征SV的调节性释放以及完整细胞中与SV相关抗原的分布非常有用。SDS凝胶电泳和免疫印迹分析表明，与未处理细胞相比，所有三个分化组中的突触素I水平都更高；而单独暴露于NGF或接受NGF和ras双重处理的细胞中，突触囊泡蛋白水平较低。文中讨论了PC12细胞中NGF和ras分化机制可能的趋同和/或分歧。
