Löffner F, Lohmann S M, Walckhoff B, Walter U, Hamprecht B
Brain Res. 1986 Jan 22;363(2):205-21. doi: 10.1016/0006-8993(86)91006-1.
Primary cell cultures derived from embryonic rat brain were characterized by immunocytochemical methods using established cell markers and monospecific antisera against cyclic nucleotide-dependent protein kinases and the synaptic vesicle protein, synapsin I. The cultures contained predominantly neurons, few astroglial cells and no oligodendroglial cells, based on immunocytochemical studies of the distribution of neuron-specific enolase, glial fibrillary acidic protein, myelin basic protein and galactocerebroside. Subsequently, the immunocytochemical localization of synapsin I, the cyclic GMP-dependent protein kinase and the various subunits of cyclic AMP-dependent protein kinase was determined. Synapsin I, a substrate for both the cyclic AMP- and Ca2+/calmodulin-dependent protein kinases, appeared particularly useful as a specific neuronal marker in primary cultures. Both immunocytochemical and immunoblotting techniques readily detected synapsin I in neuron-rich embryonic brain cultures, but indicated that synapsin I was absent from glia-rich primary cultures of newborn rat brain cells which lacked neurons. The intracellular localization of synapsin I in neurons changed markedly during the time of cell culture. In the first 10 days of cell culture, synapsin I appeared to be confined to neuronal cell bodies, whereas later it shifted to a patchy distribution in neuronal processes, perhaps indicating the transport of synapsin I in synaptic vesicles from the compartment of protein synthesis to its final synaptic location. Within neuron-rich embryonic cultures, the regulatory subunit (R-II) and the catalytic subunit (C) of cyclic AMP-dependent protein kinase appeared to be highly concentrated in neurons examined immunocytochemically. However, biochemical experiments demonstrated that R-II and C were also present in non-neuronal cell types of brain cell primary cultures. Cyclic GMP-dependent protein kinase, a marker protein for cerebellar Purkinje cells and for smooth muscle cells, was not detected immunocytochemically in neuron-rich cultures of embryonic brain cells, suggesting that Purkinje cells and smooth muscle cells were either absent from or not sufficiently developed in these cultures.
使用已确立的细胞标志物以及针对环核苷酸依赖性蛋白激酶和突触小泡蛋白突触素I的单特异性抗血清，通过免疫细胞化学方法对源自胚胎大鼠脑的原代细胞培养物进行了表征。基于对神经元特异性烯醇化酶、胶质纤维酸性蛋白、髓鞘碱性蛋白和半乳糖脑苷脂分布的免疫细胞化学研究，这些培养物主要包含神经元，少量星形胶质细胞，没有少突胶质细胞。随后，确定了突触素I、环鸟苷酸依赖性蛋白激酶和环腺苷酸依赖性蛋白激酶各亚基的免疫细胞化学定位。突触素I是环腺苷酸依赖性蛋白激酶和钙/钙调蛋白依赖性蛋白激酶的底物，在原代培养物中作为一种特异性神经元标志物显得尤为有用。免疫细胞化学和免疫印迹技术都能在富含神经元的胚胎脑培养物中轻易检测到突触素I，但表明在缺乏神经元的新生大鼠脑细胞富含胶质细胞的原代培养物中不存在突触素I。在细胞培养期间，突触素I在神经元内的定位发生了明显变化。在细胞培养的前10天，突触素I似乎局限于神经元细胞体，而后来它转移到神经元突起中的斑片状分布，这可能表明突触素I在突触小泡中从蛋白质合成部位运输到其最终的突触位置。在富含神经元的胚胎培养物中，环腺苷酸依赖性蛋白激酶的调节亚基（R-II）和催化亚基（C）在免疫细胞化学检测的神经元中似乎高度浓缩。然而，生化实验表明，R-II和C也存在于脑细胞原代培养物的非神经元细胞类型中。环鸟苷酸依赖性蛋白激酶是小脑浦肯野细胞和平滑肌细胞的标志物蛋白，在富含神经元的胚胎脑细胞培养物中未通过免疫细胞化学检测到，这表明这些培养物中要么不存在浦肯野细胞，要么其发育不充分。
