近交系小耳猪骨形态发生蛋白的骨诱导活性研究

Study on the Activity of Osteogenesis of Small-ear Porcine Bone Morphogenetic Protein

目的 :探讨近交系西双版纳小耳猪骨形态发生蛋白骨诱导活性。方法 :用改良的Urist提取法从近交系小耳猪四肢皮质骨中提取纯化BMP ,行SDS PAGE蛋白电泳检测其分子量。将BMP 2mg、5mg 2种剂量分别植入 2组小鼠右侧股部肌袋中 ,于术后 7、14和 2 8d取材 ,X线摄片后行组织学观察、碱性磷酸酶检测。结果 :( 1)SDS PAGE显示小耳猪BMP相对分子量主要集中于 66KD、48KD、3 6KD、2 7KD、2 2KD及 14KD附近 ;( 2 )X线检测实验组BMP植入区及阴性对照组均未发现有明显的高密度影 ;( 3 )ALP活性检测显示 5mg组、2mg组及阴性对照组之间有显著性差异 (P <0 .0 1) ,5mg组ALP活性高于 2mg组及阴性对照组 (P <0 .0 1)。 ( 4 )组织学观察BMP 5mg组于术后 7d有大量间充质细胞增生聚集 ,第 14d有大量软骨细胞、类骨组织及少量新骨形成 ,第 2 8d可见板层骨及骨髓。结论 :研究结果显示小耳猪骨形态发生蛋白具有肯定的骨诱导活性。

Objective: To investigate the osteogenic activity of bone morphogenetic protein (BMP) of Banna small-ear pig Methods:BMP were separated and purified from cortical bone of limbs of small-ear pig through Urist improving method and MW was determined by SDS-PAGE.36 Balb/c mice were divided into 2 groups at random,then 2mg and 5mg sepBMP were separately implanted in the right thigh muscle pouch of mouse.The specimens harvested at 7,14 and 28 days after implantation were processed for measuring bone inducing activity by histological analysis and alkaline phosphatase(ALP) assay after roentgenography.Results:(1)SepBMP contained polypeptide fragments with MW at 66×10 3KD,48×10 3KD,36×10 3KD,27×10 3KD,22×10 3KD and 14×10 3KD as shown in SDS-PAGE.(2)Roentgenographs showed no singnificant high density images at the site of implantations of all mice.(3)ALP assay showed that there was significant difference ( P<0.01 ) among three groups and the ALP activities of 5mg group was higher than that of 2mg and the contral group ( P<0.01 ).(4)Histological observation showed that in 5mg group there were a large amount of quantity of mesenchymal cells proliferated and gathered on day 7 after implantation.Cartilage formation was induced within 2 weeks and lamellar bone including bone marrow was seen within 4 weeks.Conclusion:The results of this study suggest that the sepBMP possesses certain osteoinduction properties.