吗啡备用根大鼠脊髓组织神经营养活性物质的分离纯化和生物活性测定

THE ISOLATION AND BIOACTIVE ASSAY OF NEUROTROPHIC ACTIVE SUBSTANCES DERIVED FROM SPINAL CORD OF MORPHINE SPARED ROOT RAT

目的 分离纯化吗啡备用根大鼠脊髓组织提取液 ,以期获得某些神经营养活性物质。 方法 应用SephacrylS 2 0 0HR凝胶层析、高效液相色谱 (HPLC)和组织培养等技术分离和检测神经营养活性物质。 结果 备用根大鼠脊髓组织提取液能够促进体外培养的鸡胚背根节 (DRG)神经突起的生长 ;吗啡作用的大鼠脊髓组织提取液也具有同样的作用 ,但备用根大鼠和吗啡作用的大鼠脊髓组织提取液在促神经突起生长作用方面并没有明显的差异。吗啡备用根大鼠脊髓组织提取液具有明显的神经营养活性作用。吗啡备用根大鼠脊髓组织提取液的SephacrylS 2 0 0HR凝胶层析Ⅱ峰洗脱液和Ⅳ峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE分析 ,Ⅱ峰洗脱液呈现 1条分子量约为 6 5kD的蛋白质主带 ,Ⅳ峰洗脱液的蛋白质成分较为复杂。应用HPLC对凝胶层析Ⅳ峰洗脱液作进一步的分离 ,发现HPLCA峰洗脱液能够促进DRG神经突起的生长。经SDS PAGE显示 ,A峰洗脱液的两条蛋白质主带分子量分别为 30kD和 18kD。 结论 吗啡备用根大鼠脊髓组织提取液中具有神经营养活性作用的物质可能是分子量约为 6 5kD、30kD和

Objective The extract of spinal cord tissue in morphine spared root rat was isolated and purified to look for some neurotrophic active substances. Methods Neurotrophic active substances were isolated and analysed by Sephacryl S 200 HR gel chromatography、high performance liquid chromatography(HPLC)and tissue culture,ect. Results The extract of spinal cord tissue of spared root rat could enhance the neurite growth of chick embryonal dorsal root ganglion(DRG) in vitro.There was also same effect in the extract of spinal cord tissue of morphine\|treated rat.But there was no significant difference in the extract of spinal cord tissue promoting the neurite growth between spared root rat and morphine treated rat.The extract of spinal cord tissue of morphine spared root rat had evident neurotrophic active role.The peak Ⅱ eluate and peak Ⅳ eluate obtained from the spinal cord tissue extract of morphine spared root rat through Sephacryl S 200 HR gel chromatography could promote the neurite growth of DRG.According to the analysis of SDS PAGE,the peak Ⅱ eluate showed one main protein zone with a molecular weight of 65kD and the protein composition of peak Ⅳ eluate was more complicated.The peak Ⅳ eluate of gel chromatography was then furhter isolated by HPLC.It was observed.That the peak A eluate of HPLC could promote the neurite growth of DRG. It was showed by SDS PAGE that the peak A eluate presented two main protein zones with molecular weight of 30kD and 18 kD.Conclusion\ The molecular weight of neurotrophic active substances,which were isolated from the extract of spinal cord tissue of morphine spared root rat,might be 65kD,30kD and 18kD proteins.\;[