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Effect of inducible bone morphogenetic protein 2 expression on the osteogenic differentiation of dental pulp stem cells in vitro
Bone ( IF 3.5 ) Pub Date : 2020-03-01 , DOI: 10.1016/j.bone.2019.115214
Ferenc Tóth 1 , József M Gáll 2 , József Tőzsér 3 , Csaba Hegedűs 1
Affiliation  

Bone morphogenetic protein 2 (BMP-2) is a member of the transforming growth factor-β superfamily, it is known to be a factor involved in skeletal development and capable of inducing in vitro osteogenic differentiation of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) isolated from extracted third molar teeth are an ideal resource for bone tissue engineering and regeneration applications, due to their convenient isolation, safe cryopreservation, and easy maintenance in cell cultures. The aims of this study were to deliver BMP-2 under control of the tetracycline-inducible (tet-on) promoter into dental pulp stem cells and to examine whether these BMP-2 expressing cell lines are capable of promoting osteogenic differentiation in vitro. BMP-2 gene was cloned into the lentiviral transfer plasmid pTet-IRES-EGFP and used to establish the DPSC-BMP-2 cell line. DPSC, DPSC-GFP (mock) and DPSC-BMP-2 cell lines were cultured in growth medium or osteogenic medium in the presence or absence of 100 ng/ml doxycycline. To assess differentiation, alkaline phosphatase activity, calcium accumulation and gene transcription levels of different genes involved in osteogenic differentiation (BMP-2, Runx2, alkaline phosphatase, and noggin) were measured. Doxycycline-induced BMP-2 expression induced the differentiation of DPSCs into the preosteoblastic stage but could not favor the further maturation into osteoblasts and osteocytes. We found that while Runx2 gene transcription was continuously upregulated in doxycycline-treated DPSC-BMP-2 cells, the alkaline phosphatase activity and the accumulation of minerals were reduced. As a result of the increased BMP-2 expression, the transcription level of the BMP antagonist noggin was also upregulated, and probably caused the observed effects regarding alkaline phosphatase (ALP) activity and mineral deposition. Our study shows that this system is effective in controlling transgene expression in DPSC cell line. Exploration of all known factors affecting osteogenic differentiation and their interactions is of major importance for the field of regenerative medicine. As the metabolic reaction to the upregulated transgene transcription appears to be cell line-specific, a wrongly selected target gene and/or regulation system could have adverse effects on differentiation.

中文翻译:

诱导性骨形态发生蛋白2表达对牙髓干细胞体外成骨分化的影响

骨形态发生蛋白 2 (BMP-2) 是转化生长因子-β 超家族的成员,已知它是参与骨骼发育并能够诱导间充质干细胞 (MSCs) 体外成骨分化的因子。从拔出的第三磨牙中分离的牙髓干细胞 (DPSC) 是骨组织工程和再生应用的理想资源,因为它们易于分离、安全冷冻保存,并且在细胞培养中易于维护。本研究的目的是在四环素诱导型 (tet-on) 启动子的控制下将 BMP-2 递送到牙髓干细胞中,并检查这些 BMP-2 表达细胞系是否能够促进体外成骨分化。BMP-2基因被克隆到慢病毒转移质粒pTet-IRES-EGFP中,用于建立DPSC-BMP-2细胞系。DPSC、DPSC-GFP(模拟)和 DPSC-BMP-2 细胞系在存在或不存在 100 ng/ml 强力霉素的生长培养基或成骨培养基中培养。为了评估分化,测量了参与成骨分化的不同基因(BMP-2、Runx2、碱性磷酸酶和头蛋白)的碱性磷酸酶活性、钙积累和基因转录水平。强力霉素诱导的 BMP-2 表达诱导 DPSCs 分化为前成骨细胞阶段,但不利于进一步成熟为成骨细胞和骨细胞。我们发现,虽然 Runx2 基因转录在多西环素处理的 DPSC-BMP-2 细胞中持续上调,碱性磷酸酶活性和矿物质积累减少。由于 BMP-2 表达增加,BMP 拮抗剂 noggin 的转录水平也被上调,并且可能导致观察到的关于碱性磷酸酶 (ALP) 活性和矿物质沉积的影响。我们的研究表明,该系统可有效控制 DPSC 细胞系中的转基因表达。探索影响成骨分化的所有已知因素及其相互作用对于再生医学领域具有重要意义。由于对上调转基因转录的代谢反应似乎是细胞系特异性的,错误选择的靶基因和/或调节系统可能对分化产生不利影响。BMP 拮抗剂 noggin 的转录水平也被上调,并且可能导致观察到的关于碱性磷酸酶 (ALP) 活性和矿物质沉积的影响。我们的研究表明,该系统可有效控制 DPSC 细胞系中的转基因表达。探索影响成骨分化的所有已知因素及其相互作用对于再生医学领域具有重要意义。由于对上调转基因转录的代谢反应似乎是细胞系特异性的,错误选择的靶基因和/或调节系统可能对分化产生不利影响。BMP 拮抗剂 noggin 的转录水平也被上调,并且可能导致观察到的关于碱性磷酸酶 (ALP) 活性和矿物质沉积的影响。我们的研究表明,该系统可有效控制 DPSC 细胞系中的转基因表达。探索影响成骨分化的所有已知因素及其相互作用对于再生医学领域具有重要意义。由于对上调转基因转录的代谢反应似乎是细胞系特异性的,错误选择的靶基因和/或调节系统可能对分化产生不利影响。我们的研究表明,该系统可有效控制 DPSC 细胞系中的转基因表达。探索影响成骨分化的所有已知因素及其相互作用对于再生医学领域具有重要意义。由于对上调转基因转录的代谢反应似乎是细胞系特异性的,错误选择的靶基因和/或调节系统可能对分化产生不利影响。我们的研究表明,该系统可有效控制 DPSC 细胞系中的转基因表达。探索影响成骨分化的所有已知因素及其相互作用对于再生医学领域具有重要意义。由于对上调转基因转录的代谢反应似乎是细胞系特异性的,错误选择的靶基因和/或调节系统可能对分化产生不利影响。
更新日期:2020-03-01
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