骨形成蛋白-2和牙胚细胞联合作用诱导人牙周膜干细胞表达成牙骨质细胞的表型
发布时间：2018-12-15 20:43
【摘要】：牙骨质是覆盖在牙根表面的特殊矿化组织，它借助牙周膜纤维将牙齿与牙槽骨相连，在维持牙齿结构的稳定和正常生理功能上发挥着重要作用。当牙骨质发生吸收或破坏时，其自我修复能力有限，原因在于随着牙齿的发育完成，储存在牙周组织中的成牙骨质细胞数量有限同时也不易获得。人牙周膜干细胞(Humanperiodontal ligament stem cells，hPDLSCs)具有自我增殖和多向分化潜能，是牙周组织工程的种子细胞。hPDLSCs在不同的培养环境和细胞因子的作用下，可不断增殖分化成为成牙骨质细胞、成骨细胞和成纤维细胞，修复牙周缺损，使牙周组织再生。目前已知的hPDLSCs向成牙骨质细胞分化的转录信号通路有BMP(Bonemorphogenetic protein)和Wnt/β-catenin信号等。研究显示BMP-2可促进牙槽骨、牙骨质和牙周韧带的再生，也可以诱导成牙骨质细胞的祖细胞（牙囊细胞）向成牙骨质细胞方向分化,并刺激细胞表达牙骨质标志性蛋白牙骨质附着蛋白(Cementumattachment protein, CAP)和/或牙骨质蛋白(Cementum protein1, CEMP-1),本实验希望通过建立由牙胚细胞(Tooth germ cells, TGC)和BMP-2构成的体外微环境，诱导hPDLSCs向成牙骨质细胞分化，来探究牙骨质的再生及发育机制。 实验方法 1. hPDLSCs的体外培养和鉴定 2. TGC诱导培养基的制作 3. TGC联合BMP-2体外诱导hPDLSCs，，对诱导后的细胞进行形态学观察、茜素红染色，碱性磷酸酶(Alkaline Phosphatase, ALP)活性检测以及Real-time PCR检测。 实验结果 1.利用免疫磁珠法筛选所得的hPDLSCs，间充质干细胞标志物STRO-1和血管周围细胞标志物CD146表达阳性，波形蛋白(Vimentin)表达阳性，角蛋白(Cytokeratin)表达阴性。 2.ALP活性检测显示，诱导组细胞ALP活性较对照组有显著升高(P0.001)。 3. RT-PCR检测显示诱导组成牙骨质细胞相关基因：ALP、CAP，CEMP-1及OCN的转录水平表达量增加(P0.001)。 4.茜素红染色显示，诱导组细胞表面出现大量矿化结节(P0.001)。 结论 TGC和BMP-2联合作用能够诱导hPDLSCs向成牙骨质细胞的表型分化。
[Abstract]:Cementum is a special mineralized tissue covering the root surface. It connects teeth to alveolar bone with periodontal ligament fiber and plays an important role in maintaining the stability of tooth structure and normal physiological function. When the cementum is absorbed or destroyed, its self-repair ability is limited, because with the completion of tooth development, the number of cementoblasts stored in periodontal tissue is also limited and difficult to obtain. Human periodontal ligament stem cells (Humanperiodontal ligament stem cells,hPDLSCs) have the potential of self-proliferation and multidirectional differentiation and are seed cells of periodontal tissue engineering. HPDLSCs can proliferate and differentiate into cementum cells under different culture environments and cytokines. Osteoblasts and fibroblasts repair periodontal defects and make periodontal tissue regenerate. The known transcriptional signaling pathways for hPDLSCs differentiation into cementoblast include BMP (Bonemorphogenetic protein) and Wnt/ 尾-catenin. Studies have shown that BMP-2 can promote the regeneration of alveolar bone, cementum and periodontal ligament, and induce the differentiation of cementoblast progenitor cells (dental follicle cells) into cementoblast cells. And stimulate the expression of cementum iconic protein cementum attachment protein (Cementumattachment protein, CAP) and / or cementum protein (Cementum protein1, CEMP-1). The aim of this study was to establish a new method for the expression of cementum attachment protein (Tooth germ cells,) in dental germ cells. In order to explore the mechanism of cementum regeneration and development, hPDLSCs was induced to differentiate into cementoblast cells by in vitro microenvironment composed of TGC and BMP-2. Experimental method 1. In vitro culture and identification of hPDLSCs. The preparation of TGC induction medium. The morphological changes, alizarin red staining, alkaline phosphatase (Alkaline Phosphatase, ALP) activity and Real-time PCR detection were observed by TGC combined with hPDLSCs, induced by BMP-2 in vitro. Experimental results 1. The expression of hPDLSCs, mesenchymal stem cell markers STRO-1 and CD146, vimentin (Vimentin) and keratin (Cytokeratin) were positive by immunomagnetic beads. 2.ALP activity test showed that the ALP activity in the induced group was significantly higher than that in the control group (P0. 001). 3. RT-PCR analysis showed that the transcriptional levels of ALP,CAP,CEMP-1 and OCN were increased (P0.001). 4. Alizarin red staining showed that a large number of mineralized nodules appeared on the surface of cells in the induction group (P0.001). Conclusion the combined action of TGC and BMP-2 can induce the phenotypic differentiation of hPDLSCs into cementoblasts.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781
本文编号：2381260
[Abstract]:Cementum is a special mineralized tissue covering the root surface. It connects teeth to alveolar bone with periodontal ligament fiber and plays an important role in maintaining the stability of tooth structure and normal physiological function. When the cementum is absorbed or destroyed, its self-repair ability is limited, because with the completion of tooth development, the number of cementoblasts stored in periodontal tissue is also limited and difficult to obtain. Human periodontal ligament stem cells (Humanperiodontal ligament stem cells,hPDLSCs) have the potential of self-proliferation and multidirectional differentiation and are seed cells of periodontal tissue engineering. HPDLSCs can proliferate and differentiate into cementum cells under different culture environments and cytokines. Osteoblasts and fibroblasts repair periodontal defects and make periodontal tissue regenerate. The known transcriptional signaling pathways for hPDLSCs differentiation into cementoblast include BMP (Bonemorphogenetic protein) and Wnt/ 尾-catenin. Studies have shown that BMP-2 can promote the regeneration of alveolar bone, cementum and periodontal ligament, and induce the differentiation of cementoblast progenitor cells (dental follicle cells) into cementoblast cells. And stimulate the expression of cementum iconic protein cementum attachment protein (Cementumattachment protein, CAP) and / or cementum protein (Cementum protein1, CEMP-1). The aim of this study was to establish a new method for the expression of cementum attachment protein (Tooth germ cells,) in dental germ cells. In order to explore the mechanism of cementum regeneration and development, hPDLSCs was induced to differentiate into cementoblast cells by in vitro microenvironment composed of TGC and BMP-2. Experimental method 1. In vitro culture and identification of hPDLSCs. The preparation of TGC induction medium. The morphological changes, alizarin red staining, alkaline phosphatase (Alkaline Phosphatase, ALP) activity and Real-time PCR detection were observed by TGC combined with hPDLSCs, induced by BMP-2 in vitro. Experimental results 1. The expression of hPDLSCs, mesenchymal stem cell markers STRO-1 and CD146, vimentin (Vimentin) and keratin (Cytokeratin) were positive by immunomagnetic beads. 2.ALP activity test showed that the ALP activity in the induced group was significantly higher than that in the control group (P0. 001). 3. RT-PCR analysis showed that the transcriptional levels of ALP,CAP,CEMP-1 and OCN were increased (P0.001). 4. Alizarin red staining showed that a large number of mineralized nodules appeared on the surface of cells in the induction group (P0.001). Conclusion the combined action of TGC and BMP-2 can induce the phenotypic differentiation of hPDLSCs into cementoblasts.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R781
本文编号：2381260
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