软骨生长因子的提取、纯化及生物活性测定

Extraction and purification of cartilage-derived growth factor and measurement of growth factor activity

目的 通过从鸡软骨中提取具有生物活性的软骨生长因子 (CDGF) ,为进一步进行体内研究成软骨效应及临床应用打下基础。 方法 采用氯化钠抽提法分离、制备软骨粗提物 ;用肝素 琼脂糖亲合色谱反复层析提纯软骨生长因子 ;以聚丙烯酰胺凝胶电泳和银染色分析软骨生长因子 ;采用MTT法对CDGF在培养的软骨细胞上进行活性测定。 结果 软骨生长因子能与肝素 琼脂糖紧密结合 ,亲合层析中 ,在 1 4～ 1 8mol/LNaCl缓冲液中洗脱出。通过SDS PAGE凝胶电泳和银染色检测 ,得到分子量为 185 0 0的单一条带 ,高度纯化的CDGF ,其生物活性约 1～ 2ng/ml。 结论 肝素 琼脂糖亲合色谱反复层析 ,可以从鸡软骨中得到活性较高 ,电泳显示单一条带的CDGF。

Objective To extract and purify t he cartilage derived growth factor (CDGF) from chicken sterna cartilage and mea sure its activity in vitro. Methods Chicken st erna cartilage was purchased from the local poultry farm. Extraction with sodium chloride was used for the preparation of cartilage extracts. Because CDGF has a marked affinity for heparin, recycling affinity chromatography on heparin seph arose was applied for purification of CDGF. Sodium dodecyl sulfate polyacrylami de gel electr ophoresis (SDS PAGE) and silver stain visualization were used to analyze the CDGF. Growth factor activity was measured by MTT method in the cult ure cartilage cell. Results CDGF was found to bind tightly to columns of immobilized heparin and could be eluted with concentr ation of salt in the order of 1 4 1 8 mol/L NaCl, the molecular weight of CDG F was estimated to be 18500Da by SDS PAGE.Highly purified CDGF at about 1 2 ng /ml was obtained when crude cartilage extract was applied to heparin sepharose and the growth factor activity was recycled over hoparin sepharose. [WT5” HZ]Conclusion CDGF can be obtained from chicken sterna carti lage by heparin affinity chromatography. It has a high growth activity in cultu re cartilage cell.