摘要
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.
出处
《天津医药》
CAS
北大核心
2009年第10期820-822,915,共4页
Tianjin Medical Journal
基金
国家自然科学基金资助项目(项目编号:30300171)
天津市自然科学基金重点项目(项目编号:07JCZDJC07500)
天津市高等学校科技发展基金(项目编号:20060207)
关键词
骨形态发生蛋白质类
质粒
成骨细胞
基因表达
细胞分化
bone morphogenetic proteins plasmids osteoblasts gene expression cell differentiation