人牙髓细胞与牙周韧带细胞多向分化能力的比较

目的 比较人牙髓细胞(dental pulp cells,DPC)和牙周韧带细胞(periodontal ligamentcells,PDLC)的多向分化能力,揭示其干细胞成分特征,为开展干细胞介导的生物牙根再生奠定实验基础.方法 酶消化法分离培养人DPC和PDLC,流式细胞术检测STRO-1的表达.诱导细胞成牙本质及成骨分化、成脂分化和成软骨分化,von Kossa染色、抗骨钙素(osteocalcin,OCN)和牙本质涎蛋白(dentin sialoprotein,DSP)免疫组化染色、油红O染色、阿新蓝染色、抗Ⅱ型胶原免疫组化染色以及实时荧光定量反转录聚合酶链反应(RT-PCR)等检测DPC和PDLC的多向分化.结果 DPC和PDLC体外呈克隆样生长,STRO-1阳性率分别是(16.5%±4.2%)和(11.6%±1.1%).100%的DPC和83.3%的PDLC样本可多向分化.细胞诱导分化后,OCN、牙本质涎磷蛋白(dentinsialophosphoprotein,DSPP)、过氧化物酶体激活物增生受体2(peroxisomal proliferator activated receptorgamma 2,PPARγ2)、脂蛋白脂酶(lipoprotein lipase,LPL)和Ⅱ型胶原mRNA表达上调,与诱导前相比差异均有统计学意义(P<0.001),DPC和PDLC间OCN和PPARγ2基因上调倍数的差异有统计学意义(P<0.001).结论 人DPC和PDLC的间充质干细胞比例和多向分化能力相似.     

人牙源性多能干细胞重编程前后微小RNAs差异表达谱系分析

目的 对比研究两种人牙源性多能干细胞重编程前后微小RNAs（miRNAs）差异表达,交集分析、筛选特异性miRNAs。方法 利用仙台病毒将人牙髓干细胞（DPSCs）和根尖乳头干细胞（SCAP）重编程为诱导性多潜能干细胞（iPSCs）,提取总RNA,miRNAs标记、杂交,扫描芯片、读取图像,筛选差异表达miRNAs,交集分析。结果 人DPSCs和SCAP均可重编程为iPSCs。miRNAs芯片分析结果显示人DPSCs重编程后有68个差异表达miRNAs（倍数>10）,其中37个表达上调,31个表达下调;人SCAP重编程后有107个差异表达miRNAs（倍数>10）,其中68个表达上调,39个表达下调。二者取交集,均上调的有miR-302e,下调的有miR-29b-3p、miR-181b-5p、miR-4328、miR-22-5p、miR-145-5p、miR-4324、let-7b-5p、miR-181a-5p、miR-27b-3p（倍数>10）。结论 人DPSCs和SCAP重编程为iPSCs过程中有多种miRNAs参与,多数与细胞周期、上皮-间充质转化、转化生长因子β信号通路等相关。     

Tideglusib对LPS刺激的人根尖牙乳头干细胞牙/骨向分化的影响

目的 探讨Tideglusib对LPS刺激的人根尖牙乳头干细胞(stem cells from the apical papilla, SCAPs)的牙/骨向分化的影响。方法 分离培养SCAPs,流式细胞术对SCAPs进行表面分子鉴定,检测Tideglusib对SCAPs细胞增殖是否有影响。通过碱性磷酸酶(alkaline phosphatase, ALP)活性和ALP染色筛选Tideglusib促进SCAPs表达ALP活性的最佳浓度。用大肠杆菌脂多糖(lipopolysaccharide, LPS)模拟炎性微环境刺激SCAPs。采用Western blot及实时定量聚合酶链反应(quantitative real-time polymerase chain reaction, RT-qPCR)等方法检测牙/骨向分化的相关蛋白(OSX、OCN、COL-Ⅰ、DSP、RUNX2)和基因(OSX、OCN、COL-Ⅰ、DSPP、RUNX2)表达变化。结果 CCK-8实验显示：Tideglusib浓度低于50 nmol/L时对细胞增殖无抑制作用;1 nmol/L Tideglusib处理LPS刺...     

根尖牙乳头干细胞和人脐静脉血管内皮细胞联合培养对牙髓组织成血管能力的影响

目的 探讨联合培养根尖牙乳头干细胞（stem cells from the apical papilla,SCAPs）和人脐静脉血管内皮细胞（human umbilical vein endothelial cells,HUVECs）对牙髓组织血管形成能力的影响。方法 分别使用α-MEM培养基和成骨、成脂、成神经诱导培养基处理SCAPs,采用油红O染色、茜素红染色、βIII-Tubulin免疫荧光染色,分别检测SCAPs的成脂、成骨和神经向分化能力。取单独SCAPs、HUVECs细胞以及联合培养SCAPs和HUVECs细胞进行小管形成实验,分别在3、6、9 h检测各组小管形成的长度、节点数目和结合区面积。采用SPSS16.0软件包对数据进行统计学分析。结果 染色结果显示,实验组SCAPs形成了更多的矿化结节、红色脂滴和神经样细胞。小管形成实验结果表明,联合培养组细胞可以更快地形成类血管状结构,差异显著。结论 SCAPs具有成骨、成脂和神经向诱导分化能力,联合培养SCAPs和HUVECs可以加速血管结构的形成。     

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

Objectives

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells.

Methods

Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH₂PO₄,β-glycerophosphate and l-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5 mM) and TEGDMA (0.05-0.25 mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were also evaluated.

Results

DTSCs cultures were positive for STRO-1 (7.53 ± 2.5%), CD146 (91.79 ± 5.41%), CD34 (11.87 ± 3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated.

Significance

These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.     

大鼠牙髓干细胞与牙乳头间充质细胞成牙能力的比较研究

目的:探讨采用牙髓干细胞替代牙乳头间充质细胞进行牙齿再生研究的可行性。方法:分别分离培养6周龄SD大鼠切牙牙髓干细胞(DPSCs)和出生1d的SD仔鼠切牙牙乳头间充质细胞(DPMCs),对上述2种细胞的表面标记、增殖能力、体外矿化能力以及向成牙本质细胞分化能力进行比较。结果:DPSCs与DPMCs均具有较强的增殖能力和矿化能力,体外诱导均有成牙本质细胞特异性标记物DSPP基因表达,体内移植后均能够形成牙本质样结构。结论:DPSCs是一种较为理想的牙胚间充质细胞的替代细胞,在适宜的诱导环境中能够替代DPMCs进行牙齿的再生研究。     

Erythropoietin (rhEPOa) promotes endothelial transdifferentiation of stem cells of the apical papilla (SCAP)

ObjectiveMesenchymal stem cells (MSCs) have attracted worldwide attention for their capacity to repair damaged tissue, immunosuppression, ability to differentiate into several cell types and their secretome. Earlier studies have demonstrated their angiogenic potential in vitro and in vivo. However, little is known regarding pro-angiogenic inducers of stable endothelial transdifferentiation of MSCs. Here, we employed human MSCs from the Apical Papilla (SCAP) and investigated whether recombinant human erythropoietin-alpha (rhEPOa) could act as such inducer.DesignCultured SCAP cells were exposed to rhEPOa and assessed for cell growth kinetics, viability and morphology, as well as their capacity to form capillary tubule structures in selected microenvironments. RT-PCR was used to monitor endothelial markers and activation of EPO/EPOR pathway signaling components; while gelatin zymographies to assess activation of MMP-2.ResultsrhEPOa treatment initially (48 h) accelerated cell proliferation and allowed SCAP to sprout micro-tubular structures. Morphological and biochemical differentiation was accompanied by activation of MMP-2 and upregulation of PECAM-1, VEGFR2, vWF and VE-cadherin/CDH5. SCAP expressed the cognate EPO-R, while rhEPOa-treated SCAP exhibited higher expression of molecules involved in EPO/EPOR pathway (EPOR and JAK2).ConclusionrhEPOa is capable of promoting endothelial transdifferentiation of SCAP which may be of clinical value in treating of ischemic disorders.     

血小板裂解液对人根尖牙乳头干细胞和牙周膜干细胞体内外增殖、矿化能力的影响

目的:研究血小板裂解液(Platelet Lysate,PL)在体内外对人根尖牙乳头干细胞(SCAP)和牙周膜干细胞(PDLSCs)增殖、矿化的干预效应,以期找到PL应用于这两种细胞的最佳方式。方法:在矿化诱导培养体系中按一定体积比加入PL,分别作用于体内、外复合HA-TCP支架材料三维生长的SCAP和PDLSCs,扫描电镜(SEM)以及组织学观察细胞生长、分化情况。结果:体外三维培养模式下,PL能够促进SCAP和PDLSCs的增殖以及矿化细胞外基质的形成,并利于在支架材料上形成完整的细胞膜片(cell sheet)样结构,以上效应对于PDLSCs作用更为显著。而在体内移植模式下,经50 mL/L、10 mL/L PL培养体系预培养的SCAP能形成包含成牙本质细胞样细胞、牙本质样基质和牙髓组织的牙本质牙髓复合体样结构;而PDLSCs经50 mL/LPL干预后,可分化为成牙骨质细胞样细胞,并生成牙骨质样基质、牙周膜样组织。结论:一定体积浓度比的PL能促进SCAP和PDLSCs在体内外的增殖以及成骨、成牙本质分化。     

Influence of 2-hydroxyethyl methacrylate (HEMA) exposure on angiogenic differentiation of dental pulp stem cells (DPSCs)

ObjectiveThe angiogenic differentiation of dental pulp stem cells (DPSCs) is important for tissue homeostasis and wound healing. In this study the influence of 2-hydroxyethyl methacrylate (HEMA) on angiogenic differentiation was investigated.MethodsTo evaluate HEMA effects on angiogenic differentiation, DPSCs were cultivated in angiogenic differentiation medium (ADM) in the presence or absence of non-toxic HEMA concentrations (0.1 mM and 0.5 mM). Subsequently, angiogenic differentiation was analyzed on the molecular level by qRT-PCR and protein profiler analyzes of angiogenic markers and flow cytometry of PECAM1. The influence of HEMA on angiogenic phenotypes was analyzed by cell migration and sprouting assays.ResultsTreatment with 0.5 mM HEMA during differentiation can lead to a slight reduction of angiogenic markers on mRNA level. HEMA also seems to slightly reduce the quantity of angiogenic cytokines (not significant). However, these HEMA concentrations have no detectable influence on cell migration, the abundance of PECAM1 and the formation of capillaries. Higher concentrations caused primary cytotoxic effects in angiogenic differentiation experiments conducted for longer periods than 72 h.SignificanceNon-cytotoxic HEMA concentrations seem to have a minor impact on the expression of angiogenic markers, essentially on the mRNA level, without affecting the angiogenic differentiation process itself on a detectable level.     

DLX3 mutation negatively regulates odontogenic differentiation of human dental pulp cells

ObjectivesThe purpose of this study was to investigate the role of a novel mutant DLX3 on the odontogenic differentiation of human dental pulp cells (hDPCs) in tricho-dento-osseous (TDO) syndrome.DesignhDPCs were obtained from the healthy premolars, stably-expressing wild-type DLX3 (WT), novel mutant DLX3 (Mu) and control vector (NC) cells were generated using recombinant lentiviruses. The proliferation rates of WT-hDPCs and Mu-hDPCs were measured by CCK8 assay. Odonto-differentiation of hDPCs was assessed by alkaline phosphatase (ALP) activity assay, and mineralization ability was assessed by Alizarin red staining. Odontogenic markers, including DMP-1, DSPP, Nes, ALP, and DLX5, were analyzed using real-time polymerase chain reaction (qPCR). DMP-1 and DSPP expressions were further confirmed by Western blotting.ResultsCCK8 results showed that the novel mutant DLX3 decreased the proliferation rate of hDPCs compared with wild-type DLX3. qPCR showed that the novel mutant DLX3 weakened odontogenic differentiation by downregulating the expression of odontogenic genes. These results were further confirmed by Western blotting and ALP activity assay. Additionally, Alizarin red staining showed that the novel mutant DLX3 decreased the mineralization of hDPCs compared with wild-type DLX3.ConclusionsNovel de novo mutation of DLX3 significantly decreases the proliferation rate and inhibits the odontogenic differentiation and mineralization of hDPCs, suggesting that this novel mutation of DLX3 can influence the dentinogenesis in TDO syndrome.     

组蛋白去甲基化酶KDM5A在人牙髓细胞及成牙本质细胞分化中的表达

目的探讨组蛋白赖氨酸去甲基化酶KDM5A在人牙髓细胞（hDPC）中的表达模式及成牙本质分化诱导对其表达量的影响。方法体外培养hDPC,实时荧光定量聚合酶链反应和Western blot检测第1代至第8代（P1-P8代）hDPC中KDM5A mRNA和蛋白的表达量;免疫荧光检测KDM5A在hDPC中的分布;对P3代细胞进行成牙本质分化诱导,于第7天和第14天分别检测KDM5A mRNA和蛋白的表达水平。结果体外传代培养hDPC中可检测到KDM5A的表达,KDM5A mRNA和蛋白量均呈先增加后减少的趋势;hDPC细胞质及细胞核中均表达KDM5A;成牙本质向分化诱导7和14 d,KDM5A mRNA和蛋白量高于未诱导组细胞,诱导14 d表达量高于诱导7 d（P〈0.05）。结论 hDPC表达KDM5A,矿化诱导可提高KDM5A的表达。     

外泌体诱导3D纳米纤维小管中牙髓干细胞成骨/成牙本质向分化的作用研究

目的    探究基质细胞衍生因子-1α（stromal cell-derived factor-1α,SDF-1α）诱导的根尖牙乳头干细胞来源外泌体（exosomes derived from stem cells from apical papilla,SCAP-Exo）对3D纳米纤维小管中牙髓干细胞（dental pulp stem cells,DPSCs）增殖和成骨/成牙本质向分化的影响。方法    提取SDF-1α诱导的SCAP-Exo,采用透射电镜、纳米粒子跟踪技术及Western Blot进行鉴定。将DPSCs培养于3D纳米纤维小管中,扫描电镜观察DPSCs的形态与黏附状态。使用不同质量浓度（0、50、100 μg/mL）的SCAP-Exo刺激3D纳米纤维小管中的DPSCs,分别记为对照组、50 μg/mL SCAP-Exo组和100 μg/mL SCAP-Exo组,并对细胞增殖活力和碱性磷酸酶（ALP）活性进行检测;实时RT-PCR和Western Blot分别检测成骨/成牙本质向分化相关基因的mRNA和蛋白表达水平。结果    SCAP-Exo形态呈茶托状,具有双层膜结构,平均粒径为126.4 nm,能够表达外泌体标志蛋白CD9和CD81。扫描电镜观察显示DPSCs在3D纳米纤维小管中的黏附状态良好。50 μg/mL SCAP-Exo组和100 μg/mL SCAP-Exo组DSPCs的增殖活力和ALP活性均高于对照组,且100 μg/mL SCAP-Exo组较50 μg/mL SCAP-Exo组的ALP活性升高更显著（均P < 0.05）。100 μg/mL SCAP-Exo组成骨/成牙本质向分化基因（ALP、DMP-1、BSP和OCN）的mRNA和蛋白表达水平均高于对照组（均P < 0.05）。结论    DPSCs可在3D纳米纤维小管中维持良好的增殖活力。SDF-1α诱导的SCAP-Exo可增强3D纳米纤维小管中DPSCs的增殖活力和ALP活性,以及促进其向成骨/成牙本质向分化。     

Age of the donor affects the nature of in vitro cultured human dental pulp stem cells

ObjectivesThe dental pulp stem cells (DPSCs) of six donors (three young donors aged < 19 years and three adult donors aged > 25 and < 30 years) were characterized for their stem cell marker expression and differentiation potential to study the effect of donor age on DPSCs in vitro.MethodsDPSCs were cultured in αMEM supplemented with 20% fetal calf serum (conventional conditions) or on fibronectin-coated flasks with neurobasal medium supplemented with B27, bFGF and EGF (alternative conditions). DPSCs were characterized by immunofluorescence staining to detect the neural crest/mesenchymal stem cells markers P75 and CD146, respectively. The differentiation potential was tested by the induction of DPSCs into osteogenic, adipogenic and glial lineages and then by detecting the corresponding markers osteocalcin, lipidtox and S100ß, respectively.ResultsThe DPSCs of the young donors expressed CD146 only under the conventional conditions and expressed P75 regardless of the culture conditions. However, the DPSCs of adult donors expressed CD146 only under the alternative conditions and expressed P75 only under conventional conditions. Only the DPSCs of the young donors differentiated into the glial linage. The DPSCs of the adult donors differentiated more efficiently into the adipogenic linage. Osteogenic differentiation was comparable.ConclusionDonor age affects the expression of stem cell markers and differentiation potential of DPSCs. Moreover, the effect of culture conditions on DPSCs is age dependent.     

Comparison of two digestion strategies on characteristics and differentiation potential of human dental pulp stem cells

Objective: This study aimed to compare the behavior of dental pulp stem cells (DPSCs) after isolation using solutions containing either collagenase/dispase or collagenase alone.Design: DPSCs were isolated by two digestion methods (collagenase/dispase or collagenase alone) from human third molars. Immunophenotypic features were confirmed by flow cytometry for cell markers STRO-1, cluster of differentiation (CD) 146, CD45, and collagen type-I. The proliferation potential of cells was evaluated by 5-bromo-2′-deoxyuridine (brdU) incorporation assay, and finally they were assessed for multi-lineage differentiation potential. Data were analyzed using one-way analysis of variance and independent t-tests.Results: DPSCs isolated by either method showed similar levels of STRO-1, CD45, and collagen type-I and similar incorporation of brdU (P > 0.05). However, DPSCs obtained by collagenase I/dispase treatment had significantly higher numbers of CD146⁺ cells and osteogenic and chondrogenic capacities compared to those obtained by treatment with collagenase I alone (P < 0.05). On the other hand, more STRO-1+/CD164-DPSCs were found in the collagenase alone group with higher adipogenic potential.Conclusions: Different enzyme solutions gave rise to different populations of DPSCs. Dispase enhanced isolation of CD146⁺ DPSCs probably by disrupting the basement membranes of blood vessels and releasing DPCSs embedded in the perivascular niche. Furthermore, the differentiation potential of DPSCs was influenced by the change in enzyme solution.     

人牙髓细胞向成牙本质细胞分化的蛋白质组学研究

目的 采用蛋白质组学方法分析人牙髓细胞向成牙本质细胞分化过程中细胞蛋白表达谱的改变,揭示特征蛋白在分化过程中所起的作用.方法对人牙髓细胞进行矿化诱导,提取诱导前后细胞总蛋白,双向电泳分离蛋白质,DeCyder V6.0软件确定差异蛋白点,质谱鉴定差异蛋白质.结果双向电泳确认46个差异蛋白质斑点,质谱鉴定20个蛋白斑点,差异蛋白质涉及细胞周期调节、能量代谢、信号传导等细胞生物学过程.结论蛋白质组学技术可高通量筛选与人牙髓细胞向成牙本质细胞分化相关的功能蛋白.     

犬牙髓干细胞的分离培养与鉴定

目的：分离培养犬牙髓干细胞（cDPSCs）。方法：取犬磨牙获取牙髓,采用酶消法和组织块法取原代牙髓细胞,观察计数;免疫荧光和流式法鉴定细胞蛋白表达;进行成牙本质、成脂诱导。结果：酶消法和组织块法均可获得贴壁、集落生长的梭形细胞并稳定增殖。细胞克隆形成率为（15．17％±2．79％）。流式细胞术观察：CD90（24．43％±7．10％）、STRO-1（20．67％±1．42％）、CD24（2．03％±0．06％）、CD34阴性。免疫荧光法观察：Nestin,Vimentin 表达阳性、ALP 表达弱阳性、DSP 表达阴性或微弱阳性。成牙本质诱导见矿化结节,ALP 与 DSP 阳性。成脂诱导后细胞质见大量脂滴。结论：犬健康年轻磨牙牙髓组织中可稳定分离培养出具有一定克隆能力的 cDPSCs。     

过表达BCOR基因抑制根尖牙乳头干细胞成肌分化

目的：研究B细胞淋巴瘤因子6共抑制因子（BCL6co—repressor,BCOR）对根尖牙乳头干细胞成肌分化能力的影响。方法：利用HA—BCOR逆转录病毒载体过表达BCOR进行获得性功能研究;WesternBlot检测HA—BCOR的过表达效果;重组人肿瘤生长因子β1（TGF—β1）蛋白诱导根尖牙乳头干细胞体外成肌分化;通过检测成肌分化相关基因smoothened（SMO）、smoothmuscleactinalpha2（ACTA2）和caldeson（CALDl）的表达研究根尖牙乳头干细胞体外成肌分化能力。结果：WesternBlot结果显示HA-BCOR可以在根尖牙乳头干细胞有效的过表达;过表达BCOR抑制根尖牙乳头干细胞成肌分化相关基因SMO和CALDl的表达。结论：过表达BCOR基因抑制根尖牙乳头干细胞体外成肌分化,证实BCOR是根尖牙乳头干细胞成肌分化的抑制甚因。     

矿化液对人牙髓干细胞生物学特性的影响

目的 探讨矿化液对人牙髓干细胞(DPSCs)生长特性的影响.方法 用含一定浓度的β-甘油磷酸钠、抗坏血酸和地塞米松的矿化液诱导人牙髓干细胞,通过倒置相差显微镜、透射电镜、免疫细胞化学染色、von Kossa染色方法,观察和检测矿化液诱导后细胞形态、超微结构、表型和矿化基质分泌的变化,以未诱导组为对照.结果 矿化液连续培养7d,人DPSCs细胞形态明显改变,呈牙本质样极化细胞表现,另一侧为增大的胞体;超微结构显示,诱导后的细胞体积增大,核浆比例下降,胞浆细胞器丰富;诱导前细胞不表达牙本质涎磷蛋白(DSPP)和骨钙素(OC),诱导后均表达;连续培养21d,诱导组细胞形成多个矿化结节.结论 矿化液能诱导人DPSCs向成牙本质细胞样细胞分化并产生矿化基质.本研究从细胞水平上揭示了人DPSCs向成牙本质细胞分化机理,为人DPSCs应用于牙髓损伤的修复再生提供了依据.