牙髓干细胞分化为成牙本质细胞相关标记物的研究进展

体外诱导牙髓干细胞分化为成牙本质细胞,检测牙本质涎磷蛋白、碱性磷酸酶活性等相关标记物的变化,从而鉴定其分化能力,是近年来在牙髓干细胞领域中研究的热点。本文就上述标记物的研究进展进行综述。     

体外培养牙髓细胞分化相关标记物的研究进展

体外培养的牙髓细胞在形态上同一般成纤维细胞相似,但在生物学特性上却表现出较高的分化能力。近年来的研究表明,牙髓细胞中存在未分化的间充质干细胞,属于成体干细胞,在不同的诱导条件下表现出多向分化能力。本文就牙髓细胞分化过程中的相关标记物如碱性磷酸酶活性、矿化结节以及牙本质涎磷蛋白(DSPP)等研究进展作一综述。     

小鼠胚胎干细胞向成牙本质样细胞的诱导分化

目的探讨胚胎干细胞(ES细胞)向成牙本质样细胞诱导分化的方法。方法首先通过悬浮培养的方式,将ES细胞诱导分化形成拟胚体,然后将拟胚体细胞与牙髓成纤维细胞通过Transwell培养系统共培养,研究拟胚体细胞向成牙本质样细胞的分化情况。结果RT-PCR结果显示,拟胚体细胞与牙髓成纤维细胞共培养10d后,可检测到成牙本质细胞的特征性分子——牙本质涎磷蛋白(DSPP)的表达;共培养15d后,表达有所增强。而单独培养的拟胚体细胞,则始终未检测到DSPP的表达。结论通过与牙髓成纤维细胞共培养,可以促进胚胎干细胞向成牙本质样细胞分化。     

牙髓干细胞与牙本质再生相关性的研究进展

牙髓中存在具备自我更新和多向分化潜能的牙髓干细胞.这些细胞经诱导可向成牙本质细胞分化并形成牙本质样结构,有望成为牙本质再生的种子细胞.本文就牙髓干细胞的来源、生物学特性及其在牙本质再生中的应用作一综述.     

牙髓干细胞与牙本质再生相关性的研究进展

牙髓中存在具备自我更新和多向分化潜能的牙髓干细胞。这些细胞经诱导可向成牙本质细胞分化并形成牙本质样结构，有望成为牙本质再生的种子细胞。本文就牙髓干细胞的来源、生物学特性及其在牙本质再生中的应用作一综述。     

小分子整联蛋白结合配体N-连接糖蛋白家族成员在牙髓干细胞分化过程中的作用

体外诱导牙髓干细胞向成牙本质细胞分化并观察其过程,是目前研究牙髓干细胞分化机制和寻找其分化标志的主要方法。在成牙本质细胞分化和矿化过程中,小分子整联蛋白结合配体N-糖蛋白(SBLING)家族成员发挥着重要作用。下面就SBLING家族成员牙本质涎磷蛋白、牙本质基质蛋白-1和细胞外基质磷酸糖蛋白在牙髓干细胞分化过程中的作用作一综述。     

Notch配体Delta1对人牙髓干细胞分化的影响

目的:探讨Notch配体Delta1对体外人牙髓干细胞(dentalpulpstemcells,DPSCs)向成牙本质细胞分化能力的影响。方法:利用逆转录病毒载体建立高表达人Delta1基因的人牙髓干细胞系;实验分三组,正常牙髓干细胞组、转导细胞组及混合组(正常与转导细胞比例为100∶1),分别进行体外分化诱导。倒置显微镜观测各组细胞各生长期出现的时间;VonKossa染色计数各组形成钙化结节数;Westernblot法检测各组细胞牙本质涎磷蛋白表达。结果:与正常牙髓干细胞相比,转导细胞各生长期出现时间明显提前,形成的钙化细胞结节数目显著增加,牙本质涎磷蛋白表达显著升高。结论:Delta1基因转导牙髓干细胞仍保持了体外向成牙本质细胞分化的能力;Notch-Delta1信号与分化诱导因子协同作用可促进人牙髓干细胞向成牙本质细胞分化。     

人牙髓干细胞向成牙本质细胞定向分化过程中miRNAs表达谱筛选及鉴定

目的:筛选人牙髓干细胞向成牙本质细胞定向分化过程中差异表达的microRNAs(miRNAs)并进行初步鉴定。方法:体外分离、培养和鉴定人牙髓干细胞,miRNAs芯片筛选牙髓干细胞向成牙本质细胞分化过程中差异表达的miRNAs,并通过TargetScan数据库预测靶基因,实时定量PCR法对结果进行初步鉴定。结果:人牙髓干细胞vimentin、nestin、GFAP和I型胶原4种细胞表型均表达;成脂诱导3周后细胞内有油红O染色阳性脂滴出现;成牙本质诱导可见钙结节形成;基因芯片结果显示,牙髓干细胞向成牙本质细胞分化过程中,发生2倍以上表达变化的miRNAs有6条,其中上调3条,下调3条。通过miRNA靶标预测工具预测靶基因,发现hsa-miR-633和hsa-miR-210有与牙髓干细胞分化相关的靶基因;real time-PCR验证hsa-miR-633和hsa-miR-210表达变化与基因芯片结果相符。结论:人牙髓干细胞经诱导向成牙本质细胞分化过程中miRNAs表达谱具有显著变化,为牙髓干细胞分化机制研究提供了新的提示。     

CD146在人牙髓干细胞中表达的研究

目的:研究CD146在人牙髓干细胞及其诱导分化过程中的表达情况。方法:体外培养人牙髓干细胞,免疫荧光及流式细胞术检测CD146的表达。矿化诱导人牙髓干细胞分化,检测牙本质唾蛋白的表达,从mRNA及蛋白水平检测诱导过程中CD146的表达。结果:免疫荧光及流式细胞术证明人牙髓干细胞中CD146表达阳性。使用矿化诱导液培养人牙髓干细胞,通过检测到牙本质唾蛋白的表达,证明细胞已向成牙本质细胞方向分化;在此诱导过程中,CD146在人牙髓干细胞中的表达逐渐下调。CD146在人牙髓干细胞中有较特异性的表达,有可能作为其特异性标志物。     

牙髓干细胞分化为成牙本质细胞相关标记物的研究进展

体外诱导牙髓干细胞分化为成牙夺贡细胞，检测牙本质涎磷蛋白、碱性磷酸酶活性等相关标记物的变化，从而鉴定其分化能力，是近年来在牙髓干细胞领域中研究的热点。本文就上述标记物的研究进展进行综述。     

MEPE is downregulated as dental pulp stem cells differentiate

Previous studies on dental pulp cell culture have described heterogenous mixtures of cells that differentiate into odontoblasts and form mineralized dentin. OBJECTIVE: The aim of this study was to characterize the matrix extracellular phosphoglycoprotein (MEPE) expression by dental pulp stem cells (DPSC), related to cell differentiation. DESIGN: DPSC differentiation to form mineralized nodules was characterized by Alizarin red staining and micro-Raman spectroscopy. Osteogenesis SuperArray analysis was used to broadly screen for osteogenesis-related genes altered by DPSC differentiation. Relative levels of expression of MEPE and DSP were determined by semiquantitative RT-PCR and Western blot. RESULTS: Mineral analysis showed that as DPSC differentiated, they formed a carbonated hydroxyapatite mineral. Differentiation was initially marked by upregulation by Runx2, TGFbeta-related genes, EGFR and genes involved in collagen metabolism. ALP activity first increased, as DPSCs reached confluence but later decreased when cells further differentiated three weeks after confluence. MEPE was the only marker that was downregulated as DPSCs differentiated. CONCLUSION: DPSC differentiation can be characterized by downregulation of MEPE as other markers of DPSC differentiation, such as DSP, are upregulated. Expression of MEPE related to DSP and can be used to monitor DPSC as they are used for studies of odontoblast differentiation, tissue engineering or vital pulp therapy. The downregulation of MEPE as DPSC differentiate, suggests that MEPE is an inhibitor of mineralization.     

Expression of mineralization markers in dental pulp cells

There is an increasing interest in the utility of dental pulp stem cells (DPSCs) for dentin regeneration. The mechanisms involved in DPSC differentiation remain poorly understood. The purpose of the study was to investigate the mineralization capacity of human dental pulp cells (DPCs) and identify potential markers for odontoblast differentiation. The isolated DPCs expressed mesenchymal stem-cell markers as shown by flow cytometry and could differentiate in vitro into odontogenic, adipogenic, and chondrogenic lineages. Alkaline phosphatase activity of DPCs elevated over time, with significant upregulation on day 21 in odontogenic induction. Quantitative RT-PCR revealed that osteocalcin, dentin sialophosphoprotein (DSPP), and matrix extracellular phosphoglycoprotein (MEPE) expression also increased time dependently in the induction cultures. In conclusion, we isolated DPCs with stem cell characteristics. MEPE and DSPP showed a similar regulatory pattern of DPCs mineralization. MEPE along with DSPP may be potential odontogenetic differentiation markers.     

Effects of Calcitonin Gene-related Peptide on Dental Pulp Stem Cell Viability,Proliferation, and Differentiation

IntroductionPulpitis is an inflammation of dental pulp caused by bacterial proliferation near or within pulpal tissues. In advanced stages, when the inflammation is associated with pulp necrosis, pulp preservation is dependent on dental pulp stem cells (DPSCs) that can differentiate into odontoblastlike cells and produce reparative dentin. In this study, we evaluated the influence of sensory neurons through calcitonin gene-related peptide (CGRP) on DPSC viability and proliferation and the ability of DPSCs to differentiate into mineralizing cells.MethodsCommercially available DPSCs were treated with varying doses of CGRP, and metabolic activity, viability, proliferation, and cell death were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays, trypan blue staining, 5-bromo-2'-deoxyuridine cell proliferation assay, and caspase-3 staining, respectively. DPSC differentiation was assessed with alizarin red staining and by quantifying messenger RNA expression of odontoblast makers.ResultsCGRP induced a dose-dependent decrease of DPSC metabolic activity that was prevented by the CGRP receptor antagonist CGRP 8-37. The decrease in the proportion of live cells induced by CGRP is associated with a decrease of cell proliferation but not with caspase-3–dependent apoptosis. Interestingly, dexamethasone-induced DPSC differentiation into mineralizing cells was neither inhibited nor enhanced by CGRP treatment.ConclusionsThe neuropeptide CGRP has an inhibitory effect on DPSC proliferation but does not enhance or inhibit the differentiation of DPSCs into mineralizing cells. This suggests that CGRP might negatively influence the ability of DPSCs to contribute to regenerative or tissue repair processes.     

促丝裂原激活蛋白激酶在牙髓干细胞向成牙本质细胞分化和牙髓损伤修复中的作用

通过诱导牙髓干细胞（DPSC）向成牙本质细胞方向分化,龋源性牙髓炎的治疗将不再局限于根管治疗这一临床选择,修复治疗也不再成为缺失牙治疗的唯一方案。促丝裂原激活蛋白激酶（MAPK）,尤其是P38MAPK通过直接或间接磷酸化特定的转录因子,将细胞外刺激信号转导至细胞及其核内,从而引起一系列细胞生物学反应,如细胞增殖、分化、转化和程序性死亡。骨形态发生蛋白-2、矿物三氧化物聚合体和Biodentine皆可诱导DPSC向成牙本质细胞分化,而三者正是通过MAPK信号转导通路发挥作用的。在组织工程支架诱导DPSC分化过程中,支架材料通过激活P38MAPK信号转导通路促进了DPSC的分化。此外,MAPK信号转导通路参与牙髓损伤修复中DPSC的迁移、黏附和分化,参与牙髓损伤修复中牙本质的形成。由于MAPK信号转导通路在细胞增殖、分化和生存等过程中都起着十分关键的作用,因此,深入研究其反应分子、作用底物和作用机制有着重要的理论和临床意义。     

牙髓干细胞及其在组织工程中的研究进展

牙髓干细胞是具有自我更新能力和多向分化潜能,在一定条件下可向特定的细胞类型分化,产生数个亚系的前体细胞。牙髓干细胞在组织工程和临床应用中,可达到组织和器官再造的目的,具有广阔的前景。下面就牙髓干细胞的基本生物学特性、特异性表面标记及其在组织工程中的应用作一综述。     

Differentiation of stem cells from human deciduous and permanent teeth into spiral ganglion neuron-like cells

ObjectiveStem cells from pulp tissue are a promising cell-based therapy for neurodegenerative patients based on their origin in the neural crest. The aim of this study was to differentiate and evaluate the ability of human dental pulp stem cells from permanent teeth (DPSC) and stem cells from human exfoliated deciduous teeth (SHED) to differentiate into spiral ganglion neurons.DesignAfter isolation and characterization of mesenchymal stem cell properties, DPSC and SHED were treated with the neurotrophins brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and glial cell-derived neurotrophic factor (GDNF). The differentiation was identified by immunostaining and qRT-PCR analysis of neuronal markers and measuring intracellular calcium activity.ResultsAfter 2 weeks of induction, morphological changes were observed in both DPSC and SHED. The differentiated cells expressed neuron-specific class III beta-tubulin, GATA binding protein 3 (GATA3) and tropomyosin receptor kinase B, protein markers of spiral ganglion neurons. These cells also showed upregulation of the genes encoding these proteins, namely GATA3 and neurotrophic receptor tyrosine kinase 2. Intracellular calcium dynamics that reflect neurotransmitter release were observed in differentiated DPSC and SHED.ConclusionThese results demonstrate that dental pulp stem cells from permanent and deciduous teeth can differentiate into spiral ganglion neuron-like cells.     

The efficacy of mesenchymal stem cells to regenerate and repair dental structures

OBJECTIVES: Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. METHODS: Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. RESULTS: MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. CONCLUSION: Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.     

Dissecting dentine–pulp injury and wound healing responses: consequences for regenerative endodontics

A thorough understanding of the biology of the dentine–pulp complex is essential to underpin new treatment approaches and maximize clinical impact for regenerative endodontics and minimally invasive vital pulp treatment (VPT) strategies. Following traumatic and carious injury to dentine–pulp, a complex interplay between infection, inflammation and the host defence responses will occur, which is critical to tissue outcomes. Diagnostic procedures aim to inform treatment planning; however, these remain clinically subjective and have considerable limitations. As a consequence, significant effort has focussed on identification of diagnostic biomarkers, although these are also problematic due to difficulties in identifying appropriate diagnostic fluid sources and selecting reproducible biomarkers. This is further compounded by the link between inflammation and repair as many of the molecules involved exhibit significant multifunctionality. The tertiary dentine formed in response to dental injury has been purposefully termed reactionary and reparative dentine to enable focus on associated biological processes. Whilst reactionary dentine produced in response to milder injury is generated from surviving primary odontoblasts, reparative dentine, in response to more intense injury, requires the differentiation of new odontoblast‐like cells derived from progenitor/stem cells recruited to the injury site. These two diverse processes result in very different outcomes in terms of the tertiary dentine produced and reflect the intensity rather than specific nature (nonexposure versus exposure) of the injury. The subsequent identification of the odontoblast‐like cell phenotype remains challenging due to lack of unique molecular or morphological markers. Furthermore, the cells ultimately lining the newly deposited dentine provide only a snapshot of events. The specific source and plasticity of the progenitor cells giving rise to the odontoblast‐like cell phenotype are also of significant debate. It is likely that improved characterization of tertiary dentine may better clarify the influence of cell derivation for odontoblast‐like cells and their diversity. The field of regenerative endodontics offers exciting new treatment opportunities, and to maximize outcomes, we propose that the term regenerative endodontics should embrace the repair, replacement and regeneration of dentine–pulp.     

牙髓干细胞在牙髓组织中分布的研究

目的:通过特异性标志物研究牙髓干细胞(DPSC)在牙髓组织中的分布.方法:利用间充质干细胞特异性标志物STRO-1,外胚间充质干细胞(EMSC)标志物HNK-1,通过激光共聚焦技术,对DPSC在牙髓组织中的分布进行研究.结果:STRO-1抗原阳性细胞在牙髓组织中散在分布,在血管周围较为密集分布,成牙本质细胞层无分布;HNK-1抗原阳性细胞较密集分布在血管周围,成牙本质细胞层也有表达:在血管周围有二者共表达的细胞.结论:DPSC可位于血管周围,且在牙髓组织中有散在分布,与牙髓组织中残留的未分化间充质细胞分布相似,这些血管周围细胞标志物的表达揭示血管周壁可能是DPSC的微环境.