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目的研究Cbfa1调控间充质干细胞(MSCs)分化为成骨细胞的分子机制。方法用Percoll密度梯度离心法分离获得人、大鼠和小鼠的MSCs,通过苏木精-伊红染色、细胞化学、免疫细胞化学、免疫荧光及透射电镜等方法进行鉴定。传代培养后的MSCs分别用重组真核表达载体pcD-NA3.1(+)/Cbfa1-II通过脂质体系统转染,用NorthernBlot检测三种成骨细胞特异性细胞外基质蛋白mRNA的表达情况。结果转染后人、大鼠和小鼠的MSCs都有骨钙素、骨桥接蛋白和I型胶原的表达,三者间无明显差别。结论Cbfa1在转录水平定向调控间充质干细胞分化为成骨细胞,可应用于骨科疾患的基因治疗。
Objective To study the molecular mechanism of Cbfa1 regulating mesenchymal stem cells (MSCs) into osteoblasts. Methods MSCs of human, rat and mouse were isolated by Percoll density gradient centrifugation and identified by hematoxylin-eosin staining, cytochemistry, immunocytochemistry, immunofluorescence and transmission electron microscopy. The subcultured MSCs were transfected with the recombinant eukaryotic expression vector pcD-NA3.1 (+) / Cbfa1-II respectively by lipofectamine. The expression of three osteoblast-specific extracellular matrix proteins was detected by Northern blotting . Results The MSCs of human, rat and mouse had osteocalcin, osteopontin and collagen I expression after transfection. There was no significant difference among the three groups. Conclusion Cbfa1 can regulate the differentiation of mesenchymal stem cells into osteoblasts at the transcriptional level and can be used in the gene therapy of orthopedic disorders.