巨噬细胞源性神经营养因子的初步分离

Primary Isol ation of Macrophage-derived Neurotrophic Factor

本文制备了巨噬细胞条件培养基（ＭφＣＭ），并应用快速自动比色微量分析法检测ＭφＣＭ对体外培养的生后７ｄＳＤ大鼠小脑皮质神经元的作用。结果表明，分子量大于１０ｋＤ的ＭφＣＭ对神经元（细胞密度１×１０６／ｍｌ）的作用，与对照组比较有明显的神经营养活性（Ｆ＜０．０５）；用盖玻片培养法表明，该组份有促进神经元突起生长的作用。ＭφＣＭ经ＳｅｐｈａｃｒｙｌＳ－１００－ＨＲ凝胶层析和生物活性鉴定，获得了具有神经营养活性的第二峰洗脱液。此洗脱液经ＳＤＳ－聚丙烯酰胺凝胶电泳分析，证明ＭφＣＭ中神经营养活性成份的蛋白质其分子量为３１ｋＤ－６８ｋＤ之间。

n the present study,macrophage conditioned medium(M CM) was obtained, and the effect ofM CM on the survival and activity of cultured cerebellar cortical neurons of 7-day-old Spraque-Dawleyrat was measured by a rapid automated Colorimetric microassay. The data indicated that M CM frac-tion(>10kD) posseses significant neurotrophic activity to neurons at a density of 1×105 cells per millil-itre, as compared to the control (p<0. 01).Using coverslip culture method, above M CM also showsneurite promoting effects on the neurons. Additionally,the active fraction of M CM has been furtherpurified by Sephacryle S-100-HR gel chromatography and bioassay.Neurotrophic activity was found inthe M CM sephacryle-peak Ⅱ eluate。 This M CM sephacryle peak Ⅱ eluate was analysed by SDS-PAGE, The results identify that the molecular weight of neurotrophic active protein of M CM thatcould support the neuronal survival and enhance the neuronal activity is between 31kD and 68kD.