重组人骨形态发生蛋白-7活性检测方法的建立

Development of A Method for Determination of Activity of Recombinant Human Bone Morphogenetic Protein-7

目的建立一种新的重组人骨形态发生蛋白-7(Recombinant human bone morphogenetic protein-7,rhBMP-7)活性检测方法。方法将吸收性明胶海绵与rhBMP-7混匀,冻干,环氧乙烷灭菌后,植入小鼠大腿股部肌间隙,共设5组:明胶海绵对照组及50、100、150、250μg rhBMP-7/块明胶海绵实验组,分别于术后第2、3周取材,进行HE染色及蛋白含量测定。结果 HE染色结果显示,与明胶海绵对照组相比,rhBMP-7/明胶海绵组细胞数量明显增多,且随着rhBMP-7剂量的增加及作用时间的延长而逐渐增多。蛋白含量测定结果显示,与明胶海绵对照组相比,rhBMP-7/明胶海绵组蛋白含量随着rhBMP-7剂量的增加及作用时间的延长而逐渐增加,且rhBMP-7剂量与每组蛋白含量平均值呈良好的相关性。结论将rhBMP-7复合明胶海绵植入小鼠大腿肌间隙并于第2周取材进行蛋白含量测定的方法,可用于rhBMP-7活性检测。

Objective To develop a new method for determination of activity of recombinant human bone morphogenetic protein-7（rhBMP-7）.Methods KM mice were divided into one control and four test groups randomly.The mice in control group were transplanted with gelatin sponge,while those in four test groups with gelatin sponge mixed with 50,100,150 and 250 μg of rhBMP7 respectively.The absorbable gelatin sponge was mixed well with rhBMP-7 and lyophilized,then inactivated with ethylene oxide and transplanted to the thigh muscle of mice.The gelatin sponges were taken out at weeks 2 and 3 after transplantation respectively for HE staining and determination of protein content.Results HE staining showed that the numbers of cells in test groups were significantly larger than that in control group,and increased gradually with the increasing dosage of rhBMP-7 and increasing time for treatment.Similarly,compared with that in control group,the protein contents in test groups also increased gradually with the increasing dosage of rhBMP-7 and the increasing time for treatment.Meanwhile,good correlation was observed between the dosages of rhBMP-7 and the protein contents in various groups.Conclusions The activity of rhBMP-7 may be determined by transplanting gelatin sponge mixed with rhBMP-7 into the thigh muscle of mice and taking out the gelatin sponge for determination of protein content at week 2 after transplantation.