鸡胚脊髓背角神经营养物质的HPLC纯化分析

Isolating the Neurotrophic Substance from the Dorsal Horn of the Spinal Cord in the Chicken Embryo

目的 纯化鸡胚脊髓背角内的神经营养物质。方法 采用 Sephadex G- 2 0 0凝胶过滤层析法分离鸡胚脊髓背角内的神经营养物质 ,以鸡胚背根神经节细胞培养结合 MTT法检测各洗脱峰液的神经营养活性。所得活性组分用高效液相色谱技术 (HPL C)进一步分离 ,同前用 MTT法检测各洗脱峰液的神经营养活性。结果 经层析获得三个洗脱峰 ,其中第 2洗脱峰液促进培养神经细胞生长的活性最强 (P<0 .0 1) ,提示 2峰洗脱液含有神经营养物质。 HPL C后获得 7个洗脱峰。经 MTT法测定各峰洗脱液对鸡胚 DRG神经细胞的生长活性发现 F峰有明显的神经营养活性 (P<0 .0 5 )。结论 通过分离纯化获得了有较强活性的神经营养物质。

Objective To isolate the neurotrophic substance from the dorsal horn of the spinal cord in the chicken embryo. Methods The neurotrophic substance in the dorsal horn of the spinal cord from the chicken embryo was isolated partially by gel filtration sephadex G-200. The neurotrophic activity for each part of the extracts was tested by neuronal culture of the dorsal root ganglion combined with MTT method. The extracts with neurotrophic activities were further purified by HPLC using GF-250 Column. Neurotrophic activity of substance was measured by DRG neuron culture combined with MTT method. Results Three peaks from the extracts of the embryonic spinal cord were found. The substances inducing the second peak showed apparent neurotrophic activity(P<0.01), which indicated that the solution derived from the second peak contained neurotrophic substances. The HPLC analysis of the solution demonstrated seven peaks (A, B, C, D, E, F, G), with significnt higher neurotrophic activity in the F peak solution than that of the control(P<0.05). Conclusion The substances with strong neurotrophic activities were isolated and purified.