不同成骨诱导作用下骨髓间充质干细胞的基因表达模式

目的研究骨髓间充质干细胞不同成骨分化模式的分子调控机制，为将其作为骨组织缺损基因治疗的载体细胞奠定理论基础。方法用密度梯度离心结合贴壁培养法分离纯化小鼠骨髓间充质干细胞（MSCs），传代扩增后分别用矿化培养基（100nmol／L地塞米松、10mmol／Lβ-甘油磷酸钠和50mg／mL抗坏血酸）与重组人骨形成蛋白-2（rhBMP-2，500ng／mL）进行诱导。分别于诱导后1、2周行茜素红染色鉴定钙结节形成情况；诱导后1、2和3d时用RT-PCR检测Runx2、Osx、OCN和ColⅠ的基因表达情况。结果矿化诱导组和rhBMP-2诱导组，茜素红染色均呈阳性钙结节，但矿化诱导组细胞于1周后形成矿化结节，时间早于rhBMP-2诱导组（2周后形成矿化结节）；RT-PCR显示，在矿化诱导组，Runx2在诱导的1、2、3d均没有表达，Osx在诱导后2、3d有表达，OCN和ColⅠ在诱导的1、2、3d均有表达；在rhBMP-2诱导组．Runx2和Osx于诱导的第2d开始表达，OCN和ColⅠ在诱导的1、2、3d均表达。结论与rhBMP-2相比，矿化培养基通过更为直接的信号传导方式调控着骨髓间充质干细胞向成骨细胞分化，因此矿化培养基诱导骨髓间充质干细胞形成矿化结节的能力强于rhBMP-2。

The Gene Expression Patterns of Bone-marrow Mesenchymal Stem Cells under Different Osteogenic Induction

OBJECTIVE: To investigate the molecular mechanism of osteogenetic differentiation of bone-marrow mesenchymal stem cells (BMSCs) and the probability using the BMSCs to gene therapy for bone fractures. METHODS: By gradient centrifugation and adherence to the culture plastic, the MSCs were separated and purified from mouse bone marrow. The BMSCs then were cultured and sub-cultured in the osteogenetic medium (100 nmol/L Dexamethasone, 10 mmol/L beta-glycerophosphate and 50 mg/mL ascorbic acid, osteogenic supplements, OS-medium) or the recombinant human bone morphogenetic protein-2 (rhBMP-2, 500 ng/mL) for the mineralized inductions of osteogenesis, and stained by alizarin red in inducing week 1 and 2 for the identification of calcium nodule formed. The gene expressions of Runx2, Osx, OCN, and Col I were detected by RT-PCR on day 1, 2 and 3 after doing the osteogenetic inductions. RESULTS: The BMSCs induced by OS-medium and rhBMP-2 were both of positive Ca nodules with alizarin red. However, the Ca nodule induced by OS-medium formed in 1 inducing week, but the one done by rhBMP-2 occurred in 2 inducing weeks, which meant it was a late for one week. In the OS-group, the mRNA of Runx2 could not be detected on inducing day 1, 2 and 3, but the Osx mRNA appeared on inducing day 2 and 3, and also the mRNAs of OCN and Col I could be detected in all the three inducing days. In rhBMP-2 group, the Runx2 gene expressed on inducing day 2, the Osx gene expressed on inducing day 2 and 3, the OCN and Col I genes expressed on inducing day 1, 2 and 3. CONCLUSION: The BMSCs induced by OS-medium are more likely to form bone nodules than that of rhBMP-2, because of their simpler mechanisms to differentiate into osteoblasts.