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目的:探讨人牙周膜干细胞(Periodontalligament stem cell,PDLSCs)向成骨细胞定向分化潜能,从细胞、分子和基因水平分析矿化诱导过程中细胞形态、功能变化。方法:将免疫磁珠分离的人PDLSCs矿化诱导,通过倒置显微镜观察诱导细胞形态变化,ELISA法检测诱导细胞碱性磷酸酶活性(ALPase)、RT-PCR、Western blot及免疫化学染色分析其成骨相关基因、蛋白表达变化,茜素红染色法检测其矿化结节形成情况来诱导细胞做对照。结果:矿化诱导14d,细胞形态呈成骨样短突起、多角形改变,诱导7d、14d ALPase活性显著增强。21d,诱导细胞高丰度表达骨桥素(OPN)、骨钙素(OCN)、牙骨质附着蛋白(CAP)及I型胶原(COLⅠ)mRNA,而对照组只有COLⅠmRNA弱表达。同时,Western blot及免疫化学染色均显示诱导细胞分泌骨涎蛋白(BSP)。21d,仅诱导组形成大量矿化结节。结论:人PDLSCs在矿化液诱导下可向成骨细胞分化,具有成骨细胞的形态、表型和功能特点,有望成为牙周及种植体周围骨缺损修复再生的理想的种子细胞。
OBJECTIVE: To investigate the differentiation potential of PDLCs into osteoblasts and to analyze the changes of cell morphology and function during mineralization induction at the cellular, molecular and genetic levels. Methods: Human PDLSCs isolated by immunomagnetic beads were induced by mineralization. Morphological changes were observed by inverted microscope. The alkaline phosphatase (ALPase) activity was detected by ELISA. The expression of ALPase was analyzed by RT-PCR, Western blot and immunochemical staining Bone related genes, protein expression changes, alizarin red staining to detect the formation of mineralized nodules to induce cells as a control. RESULTS: On the 14th day after mineralization, the morphology of the cells showed osteogenic protuberances and polygonal changes. The ALPase activity was significantly increased on the 7th and 14th day after induction. 21d, osteopontin (OPN), osteocalcin (OCN), cementum attachment protein (CAP) and collagen Ⅰ (COLⅠ) mRNA were highly expressed in the cells, while COL Ⅰ mRNA was only weakly expressed in the control group. Meanwhile, both Western blot and immunochemical staining showed that the induced cells secrete bone sialoprotein (BSP). 21d, only the induction group formed a large number of mineralized nodules. CONCLUSION: Human PDLSCs can differentiate into osteoblasts under the condition of mineralized fluid and have the morphological, phenotypic and functional characteristics of osteoblasts, which is expected to become an ideal seed cell for repair of bone defects around periodontal and implant.