论文部分内容阅读
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 explore the predisposing factors of motor neurons in the spinal cord, Phae electrophoresis was performed on the rat peroneal muscle extract (PMe) to isolate the 35kD immunized animals that could promote the growth of spinal cord anterior horn neurons in PMe. Cell fusion and MTT colorimetric screening to obtain two anti-rat skeletal 35kD protein monoclonal antibody. MEO, MG9.2 monoclonal antibody showed inhibition on the growth activity of anterior horn motor neurons cultured in vitro. MTT OD values were 0.023 ± 0.003,0.030 ± 0.005, ME10 inhibition effect was stronger. Compared with the OD of 0.050 ± 0.002 for promoting cell growth when 35kD protein gel alone was added, the difference was significant (ME10 P <0.01, G9P <0.05). To identify the specificity of the ME10 mAb, a neutralization assay was performed. The results showed that the inhibitory effect of ME10 on motor neuron growth was specifically neutralized by 35 kD protein, indicating that ME10 is a 3 kD protein-specific antibody. ME10 monoclonal antibody immunohistochemical reaction in the skeletal muscle cytoplasm, motor nerve endings have immunoreactive substances, indicating that 35kD protein isolated from the gastrocnemius muscle extract is induced by skeletal muscle cells of nerve-induced factor .