人乳牙牙髓干细胞和恒牙牙髓干细胞的蛋白质组学比较

目的 采用蛋白质组学方法研究人乳牙牙髓干细胞（SHED）和恒牙牙髓干细胞（DPSC）中的蛋白表达差异.方法 应用双向凝胶电泳技术分离SHED和DPSC的细胞总蛋白.通过比较两种细胞的蛋白组学图谱,确定差异表达的蛋白点,而后对差异点进行基质辅助激光解析电离飞行时间质谱分析和蛋白数据库信息检索,对差异蛋白进行功能分类.结果 建立了SHED和DPSC的蛋白质组图谱,经软件分析出45个差异蛋白点,其中26个表达上调,19个表达下调,再经质谱鉴定出48种蛋白,其生物学功能涉及细胞周期、代谢等.结论 SHED与DPSC中蛋白的差异表达体现了两种细胞在结构和功能上的异同性,为进一步研究SHED和DPSC在增殖、分化中的差异,以及牙齿相关干细胞在组织工程和再生医学研究中的应用提供参考.     

低氧环境下Notch信号通路对人牙髓干细胞成牙本质向分化的影响

目的探讨低氧环境下通过低氧诱导因子-1α（HIF-1α）调控Notch信号通路对人牙髓干细胞（HDPSC）成牙本质向分化能力的影响。 方法组织块酶消化法培养HDPSC,给予氯化钴（CoCl₂）诱导的化学性低氧环境,Western blot检测HIF-1α蛋白表达,茜素红染色检测细胞矿化结节形成能力,反转录聚合酶链反应（RT-PCR）检测Notch下游靶基因Hes1与成牙本质相关基因的表达;进一步加入Notch信号通路特异性阻断剂γ分泌酶抑制剂（GSI）,观察以上指标的变化。报告基因方法检测HIF-1α对Notch信号通路的调控作用。采用R version 3.5.3软件进行统计分析,计量资料进行正态检验,多组间比较采用单因素方差分析（方差齐）或Kruskal-Wallis H检验（方差不齐）,两两比较采用LSD-t检验（方差齐）或Mann-Whitney U检验（方差不齐）。 结果（1）CoCl₂诱导的低氧条件下HDPSC中的HIF-1α蛋白水平上调,Notch下游靶基因Hes1 mRNA相对表达量为1.46 ± 0.12,相比常氧组（1.06 ± 0.09）显著增高（t = -4.64,P = 0.012）;GSI处理阻断Notch信号通路后,低氧条件下HDPSC中HIF-1α蛋白水平下调,Hes1 mRNA相对表达量降低至0.82 ± 0.14,与处理前相比差异有统计学意义（t = 5.98,P = 0.004）;常氧条件下GSI处理后的HDPSC中HIF-1α蛋白水平也明显下调,Hes1 mRNA相对表达量（0.30 ± 0.09）相比处理前也显著降低（t = 10.08,P = 0.001）;（2）矿化诱导后的茜素红染色结果显示：低氧处理后,HDPSC细胞成牙本质分化能力下降;GSI处理后,成牙本质向分化能力增强。成骨/牙本质相关基因mRNA表达水平检测结果显示：低氧处理后,BSP、OCN和DSPP的mRNA相对表达量分别为0.53 ± 0.14、0.43 ± 0.20、0.48 ± 0.11,相比常氧组（1.21 ± 0.12、1.08 ± 0.19、1.03 ± 0.13）显著降低（t_BSP = 6.30,P_BSP = 0.003;t_OCN = 4.07,P_OCN = 0.015;t_DSPP = 5.67,P_DSPP = 0.005）;GSI处理后,低氧条件下BSP、OCN和DSPP mRNA相对表达量分别为0.99 ± 0.13、1.09 ± 0.13、0.95 ± 0.16,与处理前相比显著增高（t_BSP = -4.17,P_BSP = 0.014;t_OCN = -4.83,P_OCN = 0.012;t_DSPP = -4.30,P_DSPP = 0.017）,常氧条件下BSP、OCN和DSPP mRNA相对表达量分别为1.73 ± 0.20、1.55 ± 0.08、1.52 ± 0.14,与处理前相比显著增高（t_BSP = -3.84,P_BSP = 0.027;t_OCN = -3.99,P_OCN = 0.035;t_DSPP = -4.43,P_DSPP = 0.011）。（3）荧光素酶报告基因结果显示,HIF-1α可调控NICD启动子,使双荧光素酶相对表达活性为5.37 ± 0.12,与对照组（2.09 ± 0.15）相比显著增强（t = -28.92,P<0.001）,提示HIF-1α可能使Notch信号通路激活。 结论低氧引起HDPSC中HIF-1α升高并可能通过激活Notch信号通路抑制其成牙本质向分化能力。     

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.     

Effects of cryopreservation on the characteristics of dental pulp stem cells of intact deciduous teeth

Objectives

The aim of this study was to isolate and cultivate cells from the pulp of 7-day-cryopreserved intact deciduous human teeth and evaluate the effect of cryopreservation on dental pulp stem cell (DPSC) characteristics.

Design

Twenty-six deciduous teeth were collected and allocated in two groups: immediate cell isolation (non-cryopreserved group) and intact cryopreserved (cryopreserved group). The teeth were cryopreserved in dimethylsulfoxide solution and recovered after 7 days. The success rate of isolation, proliferation, surface markers (CD14, CD29, CD34, CD45, CD73, CD90, and HLA-DR), differentiation capacity, and morphology were evaluated.

Results

Isolation success rate was 61% and 30% for the non-cryopreserved and cryopreserved groups, respectively. There were no statistical differences between the groups for the tested surface markers. The cells in both groups were capable of differentiating into three mesenchymal lineages. No statistical differences between the groups were observed through the time course proliferation assay (0, 1, 3, 5, and 7 days); however, the mean time between isolation and the fifth passage was shorter for the non-cryopreserved group (p = 0.035). The morphology of the cells was considered altered in the cryopreserved group.

Conclusion

DPSCs were obtained from cryopreserved intact deciduous teeth without changes in the immunophenotypical characteristics and differentiation ability; however, lower culture rates, proliferation potential, and morphological alterations were observed in relation to the control group.     

乳牙牙髓干细胞的研究进展

乳牙牙髓干细胞（SHED）来源于脱落的乳牙牙髓,具有较强的增殖能力、自我更新能力、多向分化潜能,而且乳牙为生物废弃物,符合伦理要求,所以渐渐成为干细胞领域研究的新热点。本文就SHED的生物学特征、培养方法、鉴定方法、多向分化潜能及其在疾病治疗中应用的研究进展作一综述。     

小檗碱对牙髓干细胞MAPK/ERK信号通路及成牙本质向分化的影响研究

目的 探讨小檗碱对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化及p38丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶(ERK)信号通路的影响.方法 采用酶消化法提取DPSCs,将培养鉴定的DPSCs传代培养至第3～5代,用于以下实验.实验分为对照组(正常培养DPSCs)、矿化...     

改良富血小板血浆促进乳牙牙髓干细胞成骨分化作用研究

目的：体外研究改良富血小板血浆（modified platelet-rich plasma,mPRP）促进人乳牙牙髓干细胞成骨分化的作用。方法：以α-MEM作为基础培养基,分别加入1％、2％、5％、10％4种不同浓度 mPRP 或者10％胎牛血清（对照）,对第4代SHED 连续培养并诱导矿化,碱性磷酸酶试剂盒检测 ALP 活性的变化,qRT-PCR 方法检测细胞内 RUNX2和骨钙素 mRNA 含量的改变。结果：不同浓度的 mPRP 均可以促进乳牙牙髓干细胞的 ALP 活性,且浓度为2％时 A 值最高;qRT-PCR 检测显示2％ mPRP 可以上调乳牙牙髓干细胞内 RUNX2及骨钙素 mRNA 的含量。结论：一定浓度的 mPRP 对乳牙牙髓干细胞的成骨分化具有一定的促进作用。     

人乳牙牙髓干细胞和成人牙髓干细胞研究进展

近年来,成体干细胞不断地从不同的组织中被分离出来,该类细胞具有多向分化潜能、较强的增殖能力和持久的自我更新能力,具备充当组织工程种子细胞的天然优势。2000年和2003年,研究者先后从成人牙髓组织和人乳牙牙髓组织中分离出具有干细胞特征的细胞,这两种细胞的发现对牙组织工程将产生重要的意义。现就这两种成体干细胞的研究进展做一综述,并展望其应用前景。     

小型猪乳牙牙髓干细胞体外分离培养及鉴定

目的体外分离培养小型猪乳牙牙髓干细胞,并对其进行生物学鉴定。方法采用滤纸片法挑取单克隆小型猪乳牙牙髓细胞,免疫组织化学染色检测,体外比较单克隆牙髓干细胞及混合牙髓干细胞向矿化组织、脂肪细胞、及神经细胞诱导分化能力。结果分离培养的小型猪乳牙牙髓干细胞呈集落状生长,克隆形成率2．74％。波形丝蛋白、间充质于细胞表面标志STRO-1染色阳性,神经干细胞特异性标志nestin染色阳性。矿化诱导结果显示单克隆牙髓干细胞及混合牙髓干细胞,均为Von-kossa染色阳性,ATJP表达明显,两者无明著差异。单克隆牙髓干细胞及混合牙髓干细胞经IBMX、胰岛素、消炎痛和氢化可的松诱导3周后,可分化为脂肪细胞,两者成脂率均较低。单克隆乳牙牙髓干细胞向神经细胞诱导分化后免疫荧光鉴定β-tubulin III表达阳性,STRO-1表达阴性。混合牙髓干细胞无明显神经元样细胞分化。结论单克隆分离培养的小型猪乳牙牙髓干细胞具有很强的克隆形成能力及多向分化潜能,其矿化能力与混合的乳牙牙髓干细胞无明显差异。     

FAM20C在体外培养人牙髓细胞中的表达及成牙本质向分化诱导对其表达的影响

目的研究人牙髓细胞（hDPC）体外培养传代过程中FAM20C的表达模式,及对hDPC成牙本质向分化诱导后FAM20C的表达变化。 方法蛋白免疫印迹法（Western blot）检测第1代至第7代（P1 ~ P7）hDPC中FAM20C蛋白的表达量;结果采用独立样本t检验;对P3代hDPC进行成牙本质分化诱导7和14 d后,反转录聚合酶链反应与Western blot检测FAM20C的mRNA和蛋白水平变化。牙本质涎磷蛋白（DSPP）、牙本质基质蛋白1（DMP1）表达变化,FAM20C的mRNA以及蛋白表达变化采用单因素方差分析。免疫荧光法检测FAM20C在hDPC中的分布。 结果体外培养的hDPC中可检测到FAM20C表达,表达量随细胞传代先增加后减少（t_P2=-10.177,P_P2= 0.001;t_P3=-18.242,P_P3<0.001;t_P4=-19.143,P_P4<0.001;t_P5=-7.452,P_P5= 0.002;t_P6= 1.357,P_P6= 0.246;t_P7= 1.099,P_P7= 0.334）;成牙本质向分化诱导7和14 d后,FAM20C的mRNA和蛋白表达量高于未诱导组细胞（F_{FAM20C mRNA}=86.252,P_{FAM20C mRNA,7 d}<0.001,P_{FAM20C mRNA,14 d}<0.001;F_FAM20C蛋白= 85.569,P_{FAM20C蛋白,7 d}= 0.002,P_{FAM20C蛋白,14 d}<0.001）。免疫荧光结果显示,成牙本质诱导前FAM20C蛋白表达于细胞核,诱导后主要表达于细胞质,诱导14 d FAM20C在细胞质中表达量高于诱导7 d。 结论hDPC中表达FAM20C,成牙本质向分化诱导上调其表达水平,诱导后在细胞质中表达较高,提示FAM20C与hDPC的成牙本质分化相关。     

组蛋白去甲基化酶Jmjd3对牙髓干细胞成牙本质向分化的影响研究

目的研究组蛋白去甲基化酶Jmjd3对牙髓干细胞(dental pulp stem cells,DPSCs)成牙本质向分化的影响。方法原代分离培养DPSCs,用流式细胞术和茜素红染色鉴定DPSCs。体外诱导DPSCs成牙本质向分化不同时间(0、3、5、7、14 d),qRT-PCR检测Jmjd3及成牙本质细胞标志物牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、牙本质基质蛋白1(dentin matrix protein1,DMP1)的表达情况。用不同浓度(0、1、10μmol/L)的Jmjd3抑制剂GSK-J4处理DPSCs 14 d,q RT-PCR检测DSPP、DMP1的表达情况。结果原代培养的DPSCs表达间充质干细胞表面标记物CD44、CD29、CD146,体外诱导培养具有成骨分化潜能。DPSCs成牙本质向分化过程中,Jmjd3、DSPP、DMP1表达水平均上调(P<0.05),且可能具有时间依赖性。10μmol/L GSK-J4处理DPSCs可明显抑制DSPP、DMP1的表达(P<0.05)。结论Jmjd3在DPSCs成牙本质向分化过程中发挥正调节作用,且与其去甲基化酶活性相关。     

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.     

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。     

Effect of tricalcium silicate on the proliferation and odontogenic differentiation of human dental pulp cells

Introduction

This study was to investigate the effects of tricalcium silicate (Ca₃SiO₅) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.

Methods

The hDPCs were seeded in culture medium with or without Ca₃SiO₅ extract and calcium hydroxide (Ca(OH)₂) extract. Proliferation of the hDPCs was measured by methyl-thiazol-tetrazolium (MTT) assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction by using odontogenic marker genes such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP 1), osteocalcin (OC), alkaline phosphatase (ALP), and collagen type I (Col I), which were verified by ALP activity assessment, mineralization assay, and immunocytochemistry staining for dentin sialoprotein (DSP).

Results

The MTT assay showed that hDPCs cultured with Ca₃SiO₅ extract proliferated more significantly as compared with Ca(OH)₂ extract. Analysis of odontogenic marker genes indicated that Ca₃SiO₅ enhanced the expression of those genes. Moreover, the extract of Ca₃SiO₅ stimulated mineralization and increased ALP and DSP production conspicuously.

Conclusions

These results reveal that Ca₃SiO₅ can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca₃SiO₅₋based cement as a pulp-capping agent.     

丹参促进人脱落乳牙牙髓干细胞神经分化的研究

目的 探讨中药丹参对人脱落乳牙牙髓干细胞(SHED)神经分化功能的影响.方法 利用不同浓度的丹参注射液和神经诱导培养基诱导SHED分化为神经元样细胞.通过观察SHED经诱导后细胞的形态变化和采用Real-Time PCR方法检测神经元标记蛋白Nestin、早期神经元标记蛋白Ⅲ-Tubulin、神经细胞粘附因子NCAM、神经分化因子NeuroD、辅助T淋巴细胞因子TH、NEF等的表达,来鉴定神经元样细胞.结果 丹参注射液诱导后SHED胞体收缩,突起伸出,形似神经元;Real-TimePCR结果显示丹参注射液促进神经元标记蛋白Nestin、早期神经元标记蛋白Ⅲ-Tubulin、神经细胞粘附因子NCAM、神经分化因子NeuroD、NEF的表达.丹参注射液联合神经培养基诱导SHED神经分化的最佳丹参注射液浓度为50mg/ml.结论 中药丹参在一定浓度范围内可促进SHED向神经元样细胞分化.     

体外持续培养对人牙髓细胞分化能力的影响

目的：探讨人牙髓细胞在体外持续培养时，其分化能力的变化趋势。方法：体外持续培养人牙髓细胞，检测不同代次细胞的碱性磷酸酶活性、钙化能力及Ⅰ型胶原表达，并对比分析。结果：随着体外培养细胞代次的增多，在同等刺激分化条件下，人牙髓细胞的碱性磷酸酶活性整体呈下降趋势，钙化结节的数目和面积逐渐减少，Ⅰ型胶原表达合成逐渐减少。结论：人牙髓细胞在体外持续培养时，分化能力逐渐下降，可能与其含有的未分化细胞（人牙髓干细胞）数目逐渐减少有直接关系。     

Effects of calcium silicate-based cements on odonto/osteogenic differentiation potential in mesenchymal stem cells

The objective of this study was to evaluate the biological effects and odonto/osteogenic differentiation potential of Biodentine, NeoMTA Plus and TheraCal LC in tooth germ-derived stem cells (TGSCs). TGSCs were exposed to the material extracts. Biocompatibility was tested with MTS cell proliferation assay. Odonto/osteogenic differentiation was assessed with alkaline phosphatase (ALP) activity and mRNA gene expressions (RUNX2, DSPP and DMP-1). Scanning electronic microscopy/energy-dispersive X-ray (SEM/EDX) analysis and pH analysis were also performed for the materials. Data were evaluated using the one-way ANOVA and Tukey's tests. TGSCs remained viable after 7 days of incubation with all tested materials. Biodentine and NeoMTA Plus showed high ALP activity and increased expression of RUNX2, DSPP and DMP-1 compared to that of TheraCal LC. All materials can induce odonto/osteogenic differentiation of MSCs in various levels. Biocompatibility and odonto/osteogenic differentiation potential of Biodentine and NeoMTA Plus are similar and superior to that of TheraCal LC.