BMP2与BMP7嵌合表达产物可诱导成骨细胞分化

Expression Products of Chimeric BMP2 and BMP7 Induce Osteoblast Differentiation

目的:构建骨形态发生蛋白(BMP)2与BMP7嵌合表达的分泌型基因载体pcDNA3-BMP2/7,检测表达产物的成骨诱导活性。方法:聚合酶链反应(PCR)扩增BMP2与BMP7的成熟肽编码基因,利用重叠延伸PCR以柔性肽(Gly4Ser)3编码序列使两者嵌合并克隆到质粒pcDNA3/sec上,转染CHO-K1细胞筛选得到稳定克隆,以其条件培养基处理鼠胚胎成纤维细胞C3H10T1/2,通过RT-PCR研究BMP2/7嵌合表达产物的活性。结果:BMP2/7嵌合表达产物可以明显提高C3H10T1/2细胞碱性磷酸酶(Alkalinephos phatase,ALP)、骨钙素(Osteocalcin,Oc)成骨细胞表型基因以及特异性转录因子Runx2(runt-related transcription factor2)mRNA的表达(P<0.01)。结论:制备的BMP2/7嵌合表达产物能够形成异源二聚体,诱导非骨源性细胞向成骨细胞分化。

Objective： To study the osteoinductive activity of chimeric molecule of bone morphogenetic protein（BMP）2 and BMP7 expressed in mammalian cells. Methods： Sequences encoding mature peptides of BMP2 and BMP7 were separately amplified by PCR and then linked by overlap-extension PCR with a DNA sequence encoding a flexible peptide （Gly,Ser）3 between them. The chimeric DNA sequence was cloned into secretory expression plasmid pcDNA3/sec and then the recombinant plasmid pcDNA3-BMP2/7 was transfected into CHO-K1 cells. In the presence of G418,cells that stably expressed BMP2/7 were screened out. Thereafter, the conditioned culture medium of the transfected cells was collected and used to treat C3H10T1/2 cells. RT-PCR was employed to study the activity of the recombinant product in inducing osteoblast differentiation. Results： The expression products of chimeric BMP2/7 significantly enhanced the mRNA expression levels of osteoblast phenotype genes, such as alkaline phosphatase, osteocalcin and osteoblast specific transcription factor runt-related transcription factor 2 in C3H10T1/2 cells（P 〈 0.01 ）. Conclusion： The chimeric expression products of BMP2/7 are capable of forming heterodimers and thus effectively induce non-bone derived cells to differentiate into osteoblasts.