Camu W, Henderson C E
Centre de Recherche de Biochimie Macromoléculaire, UPR 8402 du CNRS/INSERM U249, Montpellier, France.
J Neurosci Methods. 1992 Aug;44(1):59-70. doi: 10.1016/0165-0270(92)90114-s.
Available methods for purifying motoneurons to homogeneity from rodent spinal cord involve retrograde labelling and fluorescence-activated cell sorting, making them costly and time consuming. Motoneurons are the only neurons within the 15-day embryonic rat spinal cord to express the p75 low-affinity NGF receptor and we show that monoclonal antibody 192-IgG, which binds to the extracellular domain of p75, selectively labels a sub-population of large multipolar ventral spinal cord neurons in vitro. We have developed a bench-top panning method for purifying these motoneurons using antibody 192-IgG. Approximately 10(5) cells/spinal cord are obtained in 2 h by this method; 95% of them express p75 in culture. They rapidly put out neurites on laminin substrata, and their survival is enhanced by extracts of skeletal muscle. Using the panning method in conjunction with centrifugation on a 6.8% metrizamide cushion, separate populations of large and small motoneurons were obtained, each containing more than 90% neurons staining with antibody 192. The large motoneurons had choline acetyltransferase activities/cell approximately 4-fold greater than those of dissociated total spinal cells and 7-fold higher than those of the small motoneurons. These methods should be of considerable use for studies on factors affecting motoneuron survival and development and for transplantation of highly purified neuronal populations.
从啮齿动物脊髓中纯化运动神经元至同质的现有方法包括逆行标记和荧光激活细胞分选，这使得它们成本高昂且耗时。运动神经元是15日龄胚胎大鼠脊髓中唯一表达p75低亲和力神经营养因子受体的神经元，并且我们表明，与p75细胞外结构域结合的单克隆抗体192-IgG在体外可选择性标记一大群大的多极腹侧脊髓神经元。我们开发了一种台式淘选法，使用抗体192-IgG来纯化这些运动神经元。通过这种方法，在2小时内可从每个脊髓获得约10⁵个细胞；其中95%在培养物中表达p75。它们能在层粘连蛋白基质上迅速长出神经突，并且骨骼肌提取物可提高它们的存活率。将淘选法与在6.8%的甲泛葡胺垫层上进行离心相结合，可获得大小运动神经元的分离群体，每个群体中用抗体192染色的神经元均超过90%。大运动神经元的胆碱乙酰转移酶活性/细胞比解离的全脊髓细胞约高4倍，比小运动神经元高7倍。这些方法对于研究影响运动神经元存活和发育的因素以及高度纯化神经元群体的移植应具有相当大的用途。
