骨髓基质细胞源性神经活性物质的提取与纯化

EXTRACTION AND PURIFICATION OF NEUROTROPHIC PROTEINS FROM BONE MARROW STROMAL CELLS

目的 分离纯化骨髓基质细胞(marrow stromal cells,MSCs)胞浆中神经营养蛋白,并验证其神经活性作用。 方法 自小鼠股骨分离培养MSCs,超声粉碎后离心并收集其上清液,超滤浓缩后收集大于10 ku浓缩蛋白液。Sephadex G- 10 0层析、高效液相色谱分析(high performance liquid chromatography,HPL C)和十二烷基硫酸钠-聚丙烯酰胺凝胶不连续电泳(sodium dodecyl sulfate- polyacrylamide gel electropheresis,SDS- PAGE)等技术分离神经活性蛋白,培养脊髓运动神经元,通过MTT法及观察细胞形态来验证其神经活性作用。 结果 MSCs胞浆上清液经SephadexG- 10 0层析后发现, 峰对神经细胞生长有促进作用。对 峰行HPL C分析发现,A峰对神经细胞生长有明显促进作用。对A峰行SDS- PAGE分析,提示为单一蛋白带,分子量为13ku。 结论 MSCs胞浆中含有神经营养蛋白,其分子量为13ku。

Objective To extract and purify the cytoplasmic neurotrophic proteins from bone marrow stromal cell and to test their neurotrophic activity. Methods Bone marrow stromal cells were collected from rat femur, after ultrasonicgrind and ultracentrifugation, the supernate was ultrafiltrated and concentrated, the proteins that molecular weight was greater than 10 ku were collected. The neurotrophic active substance was extracted and purified by Sephadex G-100 gel chromatography and high performance liquid chromatography (HPLC). Then their neurotrophic activity was tested in cultured spinal cord motoneuron with MTT method and morphous observation. Results After supernate was analyzed by Sephadex G-100 chromatography, peak Ⅱ could promote the growth of neuron. A further analysis of Peak II with HPLC showed that peak A could promote the growth of neuron. The SDS-PAGE analysis of peak A indicated that a main protein zone with molecular weight of 13 ku was obtained. Conclusion The protein of 13 ku in MSCs has neurotrophic activity.