RBX诱骨活性成分的分离与纯化

Isolation and purification of osteoinduction protein in reconstituted bone xenograft

目的 重组合异种骨 (RBX)作为一种新型植骨材料在临床治疗骨缺损、骨不连等病例中取得了显著疗效 .需进一步研究重组合异种骨中诱骨活性蛋白的相对分子质量范围 .方法 采用肝素亲和层析技术纯化 RBX中诱骨活性蛋白即骨形态发生蛋白 (BMP) ,利用小鼠肌袋实验检测其骨诱导活性 ,再用聚丙烯酰胺电泳 (SDS- PAGE)测量 RBX中诱骨活性蛋白纯化后的相对分子质量 .结果 在纯化近 2 0倍后 ,获得 8.2 3m g高度纯化的蛋白质 ,组织学观察其活性发现植入小鼠股部第 10日 ,有大量软骨细胞及软骨岛的出现 ,在局部区域还可见到条索状骨小梁出现 ,SDS- PAGE电泳发现这种蛋白质的相对分子质量为 35× 10 3,经还原后单体的相对分子质量为 2 2× 10 3.结论 重组合异种骨 (RBX)中 35× 10 3的蛋白质经还原后为 2 2× 10 3,其单独使用具有异位成骨活性 ,为 RBX的临床应用提供了理论依据 .

AIM As a new kind of bone graft material, reconstituted bone xenograft have gained much success in the repair of bone defect and bone nonunion. To study relative molecular mass of osteoinduction protein in reconstituted bone xenograft. METHODS Using heparin sepharose Cl 6B chromatography, the osteoinduction protein in reconstituted bone xenograft (bone morphogenetic protein, BMP) was purified and fractions obtained from purification were bioassayed for bone induction activity by implantation into the thigh muscles of mouse. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), the molecular mass of purified protein could be known. RESULTS Osteoinduction protein in RBX was purified for 20 fold and gained protein 8.23 mg, and in vivo by the end of 10th day, much more chondrocytes and cartilage matrix form and trabeculae can also been seen. From SDS PAGE, highly purified protein is homodiner protein with relative molecular mass of 35×10 3, and 22 ×10 3 on reduction. CONCLUSION The 35×10 3 protein in reconstituted bone xenograft was 22×10 3 on reduction, and it had bone inductive activity alone. The definition of osteoinduction protein in RBX can provide a theory foundation of clinical application of RBX.