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人牙槽骨骨膜间充质干细胞的分离及维生素D对骨膜来源细胞成骨活性的影响

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells.

作者信息

Wang Yen-Li, Hong Adrienne, Yen Tzung-Hai, Hong Hsiang-Hsi

机构信息

Chang Gung University; Department of Periodontics, Chang Gung Memorial Hospital.

California Northstate University College of Medicine.

出版信息

J Vis Exp. 2018 May 4(135):57166. doi: 10.3791/57166.

Abstract

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied. Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs. Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10, 10, 10, and 10 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

摘要

间充质干细胞(MSCs)存在于多种组织中,并且可以分化为多种细胞类型,包括成骨细胞。在MSCs的牙科来源中,骨膜是一种易于获取的组织,已被确定在其形成层中含有MSCs。然而,这一来源尚未得到广泛研究。维生素D3和1,25-(OH)2D3已被证明能刺激MSCs在体外分化为成骨细胞。此外,维生素C有助于胶原蛋白的形成和骨细胞的生长。然而,尚无研究调查维生素D3和维生素C对MSCs的影响。在此,我们提出一种从人牙槽骨膜中分离MSCs的方法,并检验1,25-(OH)2D3可能对这些细胞发挥骨诱导作用的假设。我们还研究人牙槽骨膜中MSCs的存在情况,并评估干细胞的黏附和增殖。为评估维生素C(作为对照)和不同浓度的1,25-(OH)2D3(10、10、10和10 M)改变分离的MSCs中关键mRNA生物标志物的能力,使用实时聚合酶链反应(RT-PCR)测量碱性磷酸酶(ALP)、骨唾液蛋白(BSP)、核心结合因子α-1(CBFA1)、胶原蛋白-1和骨钙素(OCN)的mRNA表达。

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