重组人骨形态发生蛋白-7体内外生物学活性研究

The Study of Biological Activity of rhBMP-7 in Vitro and Vivo

采用体外、体内两种方法同时测定原核表达重组人BMP 7(rhBMP 7)的生物学活性 ,建立rhBMP 7活性测定的标准并为推广其临床应用提供依据。在体外 ,PNPP底物比色法和MTT比色法结果表明 ,10倍、10 0倍稀释上清组与细胞共同培养 72h后碱性磷酸酶 (ALP)水平和活细胞增殖数明显高于阴性对照组 (P <0 .0 5 ) ,而与标准品无显著性差异 (P >0 .0 5 )。在体内 ,将rhBMP 7植入小鼠肌间隙后 3周 ,见软骨岛和编织骨生成 ;而将rhBMP 7与胶原载体复合后植入 ,可见大量骨小梁、骨髓腔和板层骨 ,血管和骨髓丰富 ;成骨率均为8/ 8。ALP水平和钙含量 ,rhBMP 7组与标准品组相比 ,无显著性差异 (P >0 .0 5 ) ;复合组则明显高于单独植入rhBMP 7和胶原两组 (P <0 .0 5 )。

To assay the biological activity of the recombinant human bone morphogenetic protein-7(rhBMP-7) expressed in prokaryocyte with two methods in vitro and vivo, to establish a standardized method and to provide evidence for extensive clinical practice. The activity of ALP was assayed by PNPP-substrate-chromometry technique after rhBMP-7/cell being cultured 72 hours in vitro, and the number of living cells was analyzed with MTT. The result showed that ALP level and living cells proliferation were higher in groups of dilution with 10 times and 100 times than the negative control group(P<0.05), but no significant difference from that of the rhBMP-7 positive control group(P>0.05). In vivo, after three weeks of rhBMP-7 implanted into thigh muscles of mouse, cartilage island and fibrous bone were present.And after the implatation of rhBMP-7/collagen composite, massive bone trabeculae, medullary cavity of bone, lamellar bone were observed, the blood vessel and hematopoietic marrow were plenty. the bone formation was in the ratio of 8/8. ALP level and Ca content were significant high in the group with rhBMP-7/collagen than that of two groups with rhBMP-7 and collagen alone respectively(P<0.05),no significant difference between the group with rhBMP-7 and the positive control group(P>0.05).