大鼠骨骼肌35kD运动神经诱向因子单克隆抗体的产生和鉴定

Production and Assessment of Monoclonal Antibodies Specific for the 35kD Motoneuronotrophic Factor from Rat Skeletal Muscle

为探究脊髓运动神经元诱向因予,用大鼠腓骨肌提取液(PMe)作Phast电泳,分离PMe中能促进脊髓前角神经元生长的35kD蛋白带免疫动物。细胞融合,经MTT微量比色筛选,获得2株抗大鼠骨骼35kD蛋白的单克隆抗体。命名为ME10,MG9。2株单抗对体外培养的脊髓前角运动神经元的生长活性显示抑制作用,MTT OD值分别为0.023±0.003,0.030±0.005,以ME10抑制效应更强。与单独加35kD蛋白凝胶时促进细胞生长的OD值0.050±0.002比较,差别有显著意义(ME10 P<0.01,G9P<0.05)。为鉴定ME10单抗的特异性,进行中和试验。结果发现:ME10对运动神经元生长的抑制作用被35kD蛋白特异性地中和,说明ME10是3kD蛋白特异性抗体。用ME10单抗进行免疫组化反应,在骨骼肌细胞浆内,运动神经末稍都有免疫反应阳性物质,表明从腓肠肌组织提取液中分离的35kD蛋白是由骨骼肌细胞产生的神经诱向因子。

In order to study trophic factors specific for the motoneurons of the spinal cord, the 35 kD protein band which could promote the growth of anterior horn moto-neuron was electrophoretically separated from the extract of rat peroneal muscles by Phast System gel electrophoresis. Monoclonal antibodies were raised specific to the 35 kD trophic factor. Two clones of hybridomas, namely ME10 and MG9, were selected by a MTT colorimetric microassay. The chromosome numbers for the ME10 and MG9 were 75-82 and 68-80, respectively. Most of these chromosomes were telocentric and only 1 to 2 chromosomes metacentric. The isotype of both ME10 and MG9 monoclonal antibodies was TgG2b. They exhibited growth inhibitory action on the 39kD trophic factor in motoneuron cultures as measured by the MTT microassay. The growth activities (optical densities) of motoneurons with trophic factor and ME10 or MG9 antibodies were reduced to 0.023±0.005 and 0. 030±0. 005, respectively,as compared to that of control cultures without antibody being 0.050±0. 002. The growth inhibitory action of ME10 could be specifically neutralized by excess 35kD trophic factor. This result indicated that ME10 is specific antibody for the 35kD trophic factor. Immunohistochemical localization with ME10 monoclonal antibody revealed that the cytoplasm of skeletal muscle fibre and the motor end-plate of motoneurons is immunoreactively positive. These results suggested that the 35kD trophic factor isolated from the peroneal muscles is motoneurontrophic factor produced by the skeletal muscle fibres.