牙周膜细胞的多向分化及过表达转录因子 Runx2 对增强牙周膜细胞成骨/成牙骨质相关基因表达的作用。
Multilineage differentiation of dental follicle cells and the roles of Runx2 over-expression in enhancing osteoblast/cementoblast-related gene expression in dental follicle cells.
机构信息
Department of Periodontology and Institute of Oral Biomedicine, School of Dentistry, Shandong University, Jinan, China.
出版信息
Cell Prolif. 2010 Jun;43(3):219-28. doi: 10.1111/j.1365-2184.2010.00670.x.
OBJECTIVES
Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated.
MATERIALS AND METHODS
The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full-length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real-time real time RT-PCR and in vitro mineralization assay were performed to evaluate the effects of full-length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells.
RESULTS
The above-mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real-time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full-length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full-length Runx2.
CONCLUSIONS
Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over-expression up-regulated expression levels of osteoblast/cementoblast-related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2-induced changes in DFCs were more prominent than those induced by full-length Runx2.
目的
牙周膜细胞（DFC）提供牙周组织的起源，而 Runx2 对于骨形成和牙齿发育至关重要。在本研究中，评估了 DFC 的多能性，并研究了 Runx2 对其的影响。
材料和方法
将 DFC 诱导分化为成骨细胞、脂肪细胞或软骨细胞，并用茜素红染色、油红 O 染色或阿利新蓝染色来揭示分化状态。骨髓基质细胞（BMSC）和原代小鼠成纤维细胞作为对照。还将 DFC 感染编码全长 Runx2 或缺乏 VWRPY 基序的突变型 Runx2 的重组逆转录病毒。通过 Western blot 分析、实时 RT-PCR 和体外矿化试验来评估全长 Runx2 或突变型 Runx2 对细胞成骨/成牙骨质分化的影响。
结果
上述染色方法表明，DFC 分别成功地诱导分化为成骨细胞、脂肪细胞或软骨细胞，证实了牙囊组织中存在多能间充质干细胞。然而，DFC 培养物中的染色强度比 BMSC 培养物弱。实时 PCR 分析表明，突变型 Runx2 诱导 OC、OPN、Col I 和 CP23 的表达水平比全长 Runx2 更显著增加。矿化试验还表明，突变型 Runx2 比全长 Runx2 更显著地增加了矿化结节的形成。
结论
多能 DFC 可在体外诱导分化为成骨细胞、脂肪细胞或软骨细胞。Runx2 过表达上调成骨细胞/成牙骨质相关基因的表达水平，并增强 DFC 的体外成骨分化。此外，突变型 Runx2 诱导的 DFC 变化比全长 Runx2 更显著。
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