牙髓干细胞分化过程中L型钙离子通道羧基末端的表达

目的:探讨牙髓干细胞(DPSCs)分化过程中L型钙离子通道羧基末端的表达。方法:利用酶消化法体外分离、培养大鼠牙髓干细胞;吉姆萨染色法检测大鼠牙髓干细胞的克隆形成能力;神经诱导体系下诱导牙髓干细胞向神经样细胞分化,免疫荧光染色检测细胞分化后胶质纤维酸蛋白(glial fibrillary acidic pro-tein,GFAP)的表达和细胞分化前后L型钙离子通道Cav 1.2及羧基末端的表达。结果:牙髓干细胞的克隆形成能力为每1 000个细胞形成2～17个克隆;免疫荧光染色检测诱导后细胞GFAP表达阳性;免疫荧光染色检测显示:牙髓干细胞分化前L型钙离子通道Cav 1.2羧基末端表达于细胞膜上,细胞分化后羧基末端同时表达于细胞膜上和细胞核中。结论:L型钙离子通道Cav 1.2羧基末端在牙髓干细胞分化过程中发生核转位,羧基末端可能在牙髓干细胞的分化过程中发挥着一定的作用。     

Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo

IntroductionSubstance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis.MethodsDental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining.ResultsSP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10^?8 M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo.ConclusionsSP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.     

Stem cell‐based tooth and periodontal regeneration

Currently regeneration of tooth and periodontal damage still remains great challenge. Stem cell‐based tissue engineering raised novel therapeutic strategies for tooth and periodontal repair. Stem cells for tooth and periodontal regeneration include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from the dental apical papilla (SCAPs), and stem cells from human exfoliated deciduous teeth (SHEDs), dental follicle stem cells (DFSCs), dental epithelial stem cells (DESCs), bone marrow mesenchymal stem cells (BMMSCs), adipose‐derived stem cells (ADSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). To date, substantial advances have been made in stem cell‐based tooth and periodontal regeneration, including dentin–pulp, whole tooth, bioroot and periodontal regeneration. Translational investigations have been performed such as dental stem cell banking and clinical trials. In this review, we present strategies for stem cell‐based tissue engineering for tooth and periodontal repair, and the translational studies.     

炎症微环境下芦丁对牙周膜干细胞成骨分化能力的影响

目的 研究芦丁（rutin）对炎症微环境下牙周膜干细胞成骨分化能力的影响。方法 采用有限稀释法分离纯化获得牙周膜干细胞,采用流式细胞术鉴定牙周膜干细胞。以脂多糖（lipopolysaccharide, LPS）刺激牙周膜干细胞,建立体外炎症模型。实验分为4组,第1组使用α-MEM培养基培养牙周膜干细胞,第2组使用含有脂多糖的α-MEM培养基培养牙周膜干细胞,第3组在含有脂多糖的α-MEM培养基加入芦丁培养牙周膜干细胞,第4组使用含有芦丁的α-MEM培养基培养牙周膜干细胞。通过碱性磷酸酶染色、碱性磷酸酶活性测试、茜素红染色、RT-PCR以及蛋白免疫印迹等方法检测成骨分化能力的改变。采用SPSS 17.0软件包对数据进行统计分析。结果 CCK-8和碱性磷酸酶活性测试结果显示,10 μmol/L芦丁对炎症状态下牙周干细胞增殖和成骨分化作用最明显。碱性磷酸酶染色和茜素红染色结果显示,10 μmol/L芦丁可以改善炎症微环境下牙周膜干细胞的成骨分化能力。RT-PCR、蛋白免疫印迹结果显示,芦丁可以增强炎症状态下COL1、ALP、RUNX2等成骨基因和成骨蛋白的表达。结论 芦丁可以增强炎症微环境下牙周膜干细胞的成骨分化能力。     

Comparative proteomic profiling of human dental pulp stem cells and periodontal ligament stem cells under in vitro osteogenic induction

ObjectiveThis study aimed to compare the proteomic profiling of human dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) under in vitro osteogenic induction, which imitates the microenvironment during osteo-/odontogenesis of DPSCs and PDLSCs.DesignThe proteomic profiles of osteoinduced DPSCs and PDLSCs from a single donor were compared using the isobaric tag for relative and absolute quantitation (iTRAQ) technique and subsequent bioinformatics analysis.ResultsA total of 159 differentially expressed proteins in PDLSCs and DPSCs were identified, 82 of which had a higher expression level in PDLSCs, while 77 were more highly expressed in DPSCs. Among these enriched proteins, certain members from the collagen, heat shock protein and protein S100 families may distinguish osteoinduced PDLSCs and DPSCs. Gene ontology (GO) classification revealed that a large number of the enriched terms distinguishing PDLSCs and DPSCs are involved in catalytic activity, protein binding, regulation of protein metabolic processes and response to stimulus. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated several involved pathways, including the fatty acid biosynthesis pathway, pantothenate and CoA biosynthesis pathway, arachidonic acid metabolism pathway and PPAR signaling pathway. Further verification showed that the mineralization and migration capacities of PDLSCs were greater than those of DPSCs, in which heat shock protein beta-1, Protein S100-A10 and S100-A11 may play a part.ConclusionsLess than 5% of the differentially expressed proteins make up the comparative proteomic profile between osteoinduced PDLSCs and DPSCs. This study helps to characterize the differences between osteoinduced PDLSCs and DPSCs in vitro.     

Inhibition of human dental pulp stem cell differentiation by Notch signaling

Notch signaling plays a critical role in development and cell fate specification. Notch receptors and ligands have been found to be expressed in dental epithelium or mesenchyme in the developing tooth, suggesting that Notch signaling may regulate odontogenesis. Post-natal human dental pulp stem cells (DPSCs) isolated from the dental pulp have characteristics of mesenchymal stem cells and can differentiate into odontoblasts. In this study, we examined whether Notch signaling regulated the odontoblastic differentiation of DPSCs. We found that over-expression of the Notch ligand, Jagged-1, activated the Notch signaling pathway in DPSCs. Jagged-1 inhibited the odontoblastic differentiation of DPSCs in vitro. Jagged-1-expressing DPSCs could not form mineralized tissues in vivo. Moreover, over-expression of the constitutively activated Notch1 intracellular domain (Notch-ICD) also inhibited odontoblastic differentiation of DPSCs. Taken together, our results demonstrate that Notch signaling can inhibit the odontoblastic differentiation of DPSCs.     

Apical tooth germ cell-conditioned medium enhances the differentiation of periodontal ligament stem cells into cementum/periodontal ligament-like tissues

Background and Objective: Limitations of current periodontal regeneration modalities in both predictability and extent of healing response, especially on new cementum and attachment formation, underscore the importance of restoring or providing a microenvironment that is capable of promoting the differentiatiation of periodontal ligament stem cells (PDLSCs) towards cementoblast‐like cells and the formation of cementum/periodontal ligament‐like tissues. The aim of this study was to investigate the biological effect of conditioned medium from developing apical tooth germ cells (APTG‐CM) on the differentiation and cementogenesis of PDLSCs both in vitro and in vivo. Material and Methods: Using the limiting dilution technique, single‐colony‐derived human PDLSCs were isolated and expanded to obtain homogeneous populations of PDLSCs. Morphological appearance, cell cycle analysis, bromodeoxyuridine incorporation, alkaline phosphatase (ALP) activity, mineralization behavior, gene expression of cementoblast phenotype and in vivo differentiation capacities of PDLSCs co‐cultured with APTG‐CM were evaluated. Results: The induced PDLSCs exhibited several characteristics of cementoblast lineages, as indicated by the morphological changes, increased proliferation, high ALP activity, and the expression of cementum‐related genes and calcified nodule formation in vitro. When transplanted into immunocompromised mice, the induced PDLSCs showed tissue‐regenerative capacity to produce cementum/periodontal ligament‐like structures, characterized by a layer of cementum‐like mineralized tissues and associated periodontal ligament‐like collagen fibers connecting with the newly formed cementum‐like deposits, whereas control, untreated PDLSCs transplants mainly formed connective tissues. Conclusion: Our findings suggest that APTG‐CM is able to provide a cementogenic microenvironment and induce differentiation of PDLSCs along the cementoblastic lineage. This has important implications for periodontal engineering.     

牙髓干细胞的研究

牙髓干细胞是存在于牙髓组织中的一种成体干细胞，具有高度增殖、自我更新的能力和多向分化的潜能。牙髓干细胞的研究对牙组织工程和牙齿的再生将产生重要的意义。本文就牙髓干细胞的研究现状作一综述，并对其应用前景以及目前存在的问题进行讨论。     

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.     

2型糖尿病伴牙周炎患者牙周膜干细胞骨向分化研究

目的:比较牙周炎和2型糖尿病伴牙周炎患者牙周膜干细胞成骨分化能力。方法:体外组织块法和有限稀释法克隆化培养牙周炎患者牙周膜干细胞(P-PDLSCs组)和2型糖尿病伴牙周炎患者牙周膜干细胞(D-PDLSCs组),计算克隆形成率,免疫荧光检测细胞表型分子CD146、STRO-1进行干细胞鉴定,矿化诱导后茜素红染色观察矿化结节形成,实时定量聚合酶链反应(real time PCR)检测成骨相关基因表达。结果:P-PDLSCs组和D-PDLSCs组细胞的克隆形成率分别为(25.6±2.7)%和(17.9±1.7)%(P﹤0.05);P-PDLSCs组CD146和STRO-1表达明显高于D-PDLSCs组;成骨诱导21 d后茜素红染色,2组均出现不同程度的矿化结节,P-PDLSCs组的矿化能力较D-PDLSCs组强;成骨诱导1周后D-PDLSCs组碱性磷酸酶(alkaline phosphatase,ALP)、Runx-2和Ⅰ型胶原蛋白(type-Ⅰcollagen,Col-Ⅰ)的mRNA表达均明显低于P-PDLSCs组(P<0.05)。结论:糖尿病伴牙周炎患者牙周膜干细胞成骨分化能力低于单纯牙周炎患者牙周膜干细胞。     

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目的 比较微小RNA（miRNAs）在人牙髓干细胞（dental pulp stem cells, DPSCs）及非DPSCs中的表达差异,探讨其在维持DPSCs干性状态中的作用。方法 本研究于2013年1—10月在福建医科大学附属口腔医院完成。 原代培养人牙髓细胞,利用结合了STRO-1特异性抗体的免疫磁珠分选获得DPSCs,并进行成牙本质样细胞的诱导分化,检测碱性磷酸酶（ALP）、骨钙素（OC）值以及进行von Kossa染色,鉴定其分化能力。采用miRNA基因芯片技术,检测DPSCs和非DPSCs中miRNAs的表达,筛选出差异表达的miRNAs。结果 与非DPSCs相比,DPSCs中表达上调超过2倍的miRNAs有11个,表达下调超过2倍的miRNAs有3个。结论 miRNAs表达谱的变化可能与DPSCs干性状态的维持相关。     

Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro

IntroductionDental pulp stem cells (DPSC) are very attractive in regenerative medicine. In this study, we focused on the characterization of the functional properties of mesenchymal stem cells derived from DPSCs. Currently, it is unknown whether inflammatory conditions present in an inflamed dental pulp tissue could alter the immunomodulatory properties of DPSCs. This study aimed to evaluate the immunomodulatory capacity in vitro of DPSCs derived from healthy and inflamed dental pulp.MethodsDPSCs from 10 healthy and inflamed dental pulps (irreversible pulpitis) were characterized according to the minimal criteria of the International Society for Cell Therapy, proliferation, differential potential, and colony-forming units. Furthermore, the immunomodulatory capacity of DPSCs was tested on the proliferation of T lymphocytes by flow cytometry and the in vitro enzyme activity of indoleamine 2, 3-dioxygenase.ResultsThere were no significant differences in the DPSC characteristics and properties such as immunophenotype, tridifferentiation, colony-forming units, and proliferation of the DPSCs derived from normal and inflamed pulp tissue. Furthermore, there were significant differences in the immunomodulatory capacity of DPSCs obtained from human healthy dental pulp and with the diagnosis of irreversible pulpitis.ConclusionsOur results showed that DPSCs isolated from inflamed dental pulp showed typical characteristics of MSCs and diminished immunosuppressive capacity in vitro in comparison with MSCs derived from healthy dental pulp. Further investigation in vivo is needed to clarify the mechanism of this diminished immunosuppressive capacity.     

Isolation and characterization of dental pulp stem cells from a supernumerary tooth

Background: Dental pulp stem cells (DPSCs) were primarily derived from the pulp tissues of primary incisors and permanent third molar teeth, whereas no report to our knowledge has yet been documented on deriving DPSCs from the other tooth types. The aim of this study is to present a novel approach of harvesting stem cells from a supernumerary tooth (a mesiodens). Materials and methods: The pulp tissues from a mesiodens of a 20‐year‐old healthy male patient and the left lower deciduous canine of a healthy 10‐year‐old boy (the positive control) were extracted and cultured for DPSCs, which were examined with stem cells (Oct‐4, Nanog and Rex‐1) and differentiation (Osteonectin and Nestin) markers. Furthermore, DPSCs were directionally differentiated to osteogenic and adipogenic cell lineages. Results: Dental pulp stem cells derived from the mesiodens were capable of differentiating into adipogenic and osteogenic lineages. The mesioden’s DPSCs also expressed stem cell and differentiation markers, which suggested their stem cell origin and differentiation capability. All the aforementioned results for the mesiodens were consistent with those of the DPSCs derived from the positive control. Conclusion: We have demonstrated the feasibility of deriving DPSCs from a usually discarded tissue such as a supernumerary tooth.     

人牙髓干细胞及非牙髓干细胞中微小RNA表达谱比较研究

目的 比较微小RNA（miRNAs）在人牙髓干细胞（dental pulp stem cells, DPSCs）及非DPSCs中的表达差异,探讨其在维持DPSCs干性状态中的作用。方法 本研究于2013年1—10月在福建医科大学附属口腔医院完成。 原代培养人牙髓细胞,利用结合了STRO-1特异性抗体的免疫磁珠分选获得DPSCs,并进行成牙本质样细胞的诱导分化,检测碱性磷酸酶（ALP）、骨钙素（OC）值以及进行von Kossa染色,鉴定其分化能力。采用miRNA基因芯片技术,检测DPSCs和非DPSCs中miRNAs的表达,筛选出差异表达的miRNAs。结果 与非DPSCs相比,DPSCs中表达上调超过2倍的miRNAs有11个,表达下调超过2倍的miRNAs有3个。结论 miRNAs表达谱的变化可能与DPSCs干性状态的维持相关。     

SHH和bFGF体外诱导人牙髓干细胞向神经细胞分化的研究

目的：研究体外使用音猬因子（SHH）、碱性成纤维生长因子（bFGF）体外诱导人牙髓干细胞（DPSCs）分化为神经细胞的可行性,以优化人牙髓干细胞向神经细胞分化的诱导条件。方法：从因正畸或阻生拔除的第一前磨牙或第三磨牙中提取牙髓,采用酶消化及过滤法得到单细胞悬液,有限稀释法培养分离的原代人牙髓干细胞,并进行克隆化培养,检测间充质干细胞特异性标志物STRO-1的表达。将人牙髓干细胞分别接种于含有不同浓度诱导液,MTT法检测不同时间、两种因子单独或联合对细胞增殖能力的影;免疫荧光法检测抗微管相关蛋白（MAP-2）、神经元烯醇化酶（NSE）、胶质原纤维酸性蛋白（GFAP）的表达。透射电镜观察诱导前后细胞超微结构。结果：克隆来源细胞的间充质干细胞特异性标志物STRO-1表达阳性。100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF单独作用促增殖作用最强（P〈0.05）,碱性成纤维生长因子bFGF单独作用各组及对照组均未检测出神经元样细胞。音猬因子SHH作用各组检测到阳性细胞。而100μg/L音猬因子SHH与20μg/L碱性成纤维生长因子bFGF联合增殖和分化作用均优于其它组。透射电镜观察到神经元样细胞表现。结论：100μg/L音猬因子和20μg/L碱性成纤维生长因子联合可以在体外有效诱导人牙髓干细胞分化为神经细胞。     

人恒牙牙髓干细胞分化为脂肪细胞的体外实验研究

目的验证人恒牙牙髓组织来源的牙髓干细胞在体外向脂肪细胞的定向分化,并对分化后的细胞进行鉴定。方法从正畸治疗减数拔除的恒前磨牙中分离牙髓组织,应用酶消化法获得牙髓细胞。单抗Stro-1标记、免疫磁珠阳性分选系统分选获得牙髓干细胞,第3代牙髓干细胞用成脂肪向诱导培养基向脂肪细胞诱导分化。用油红O染色鉴定成脂肪向分化,RT-PCR检测脂肪细胞的特异相关或标志基因。以同期培养的未诱导的普通培养基培养的DPSCs做阴性对照;以同期培养的骨髓间充质干细胞成脂肪向分化的结果做阳性对照。结果人恒牙牙髓干细胞经成脂肪向培养基诱导后表现出脂肪细胞特性,油红O染色结果为阳性,RT-PCR检测成脂肪向分化相关基因过氧化物酶增殖物激活受体γ2、脂肪酶结合蛋白aP2和脂蛋白脂酶均有阳性表达。结论人恒牙牙髓干细胞在体外具有分化为脂肪细胞的潜能。     

瘦素对人牙髓干细胞增殖及分化的影响

目的 探讨瘦素(leptin)对体外培养的牙髓干细胞增殖、分化的影响.方法 体外培养牙髓干细胞,采用MTT法及流式细胞技术检测不同浓度瘦素对牙髓干细胞增殖作用的影响,通过碱性磷酸酶、Yon Kossa染色及矿化相关蛋白(DSP、OCN)的免疫组化染色来检测瘦素对牙髓干细胞分化的影响.结果 瘦素对牙髓干细胞增殖没有显著作用,但是对牙髓干细胞的ALP活性呈浓度依赖性促进.Von Kossa染色可见矿化结节形成,DSP及OCN表达阳性.结论 瘦素可促进牙髓干细胞向成牙本质细胞分化.