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。     

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.     

釉质基质蛋白通过miR-32对乳牙牙髓干细胞成骨与成脂分化的影响

目的:探讨釉质基质蛋白(EMPs)对乳牙牙髓干细胞(SHED)成骨和成脂作用的影响,并探讨其分子机制.方法:采用流式细胞术检测SHED表面抗原CD73、CD146、CD34和CD45的表达.通过OB成骨诱导液诱导SHED,采用茜素红染色检测其成骨分化能力.将SHED分为4组,NC组为无效序列shRNA干扰SHED,EM...     

人乳牙牙髓干细胞在干细胞治疗中的应用

乳牙牙髓干细胞（SHED）是牙源性干细胞的一种,属外胚间充质干细胞。作为一种理想的干细胞来源,SHED在干细胞治疗中有良好的应用前景。本文阐述了SHED的生物学特征及其在干细胞治疗中的优势,探讨了SHED在组织再生和修复中发挥的多向分化潜能、细胞分泌功能和免疫调节功能等方面的功能作用。此外,本文还介绍了SHED在各系统、器官疾病治疗中的临床应用,重点阐述了用SHED进行干细胞移植在牙髓—牙本质再生、颌骨再生、神经系统疾病治疗和免疫系统疾病治疗方面的研究进展。     

体外连续扩增对乳牙源牙髓干细胞生物学特性的影响

目的 :研究人脱落乳牙牙源髓干细胞(SHED)在体外长时期扩增,培养至20代后生物学特性的改变,探讨长时间扩增培养对SHED特性的影响。方法:从健康儿童脱落的乳牙中分离牙髓干细胞,在常规条件下扩增培养至第20代,比较第4代、第20代SHED,在细胞形态、增殖速率、多向分化、细胞凋亡等方面的差异。结果:与第4代相比,第20代细胞增殖速率减慢,成脂、成骨分化能力减弱,凋亡细胞增多,但仍能保持干细胞特有的免疫表型,高表达CD73、CD90、CD105,低表达CD34、CD11b、CD19、CD45和HLA-DR。结论 :随着体外扩增至20代,SHED虽能维持干细胞特有的免疫表型,但其生物学特性发生改变,已成为衰老细胞,不再适用于组织工程及干细胞治疗。     

釉质基质蛋白对乳牙牙髓干细胞体外增殖、分化能力的影响

目的：研究釉质基质蛋白（enamel matrix proteins,EMPs）对人脱落乳牙牙髓干细胞（stem cells from hu—man exfoliated dediduous teeth,SHED）体外增殖分化能力的影响。方法：利用酶消化法联合组织块法获得脱落乳牙牙髓干细胞,并进行形态学观察。三氯乙酸法制备EMPs,用不同浓度的EMPs对SHED进行诱导,利用四唑盐比色法（MTT）检测并分析诱导后的SHED增殖活性的变化,检测经诱导后的培养液中碱性磷酸酶（ALP）。RT—PCR检测牙本质涎磷蛋白（dentin sialophosphoprotein,DSPP）及牙本质基质蛋白1（dentin matrix protein1,DMP-1）的mRNA表达。结果：人脱落乳牙牙髓干细胞呈集落生长,并且在体外具有一定的自我增殖能力。EMPs对乳牙牙髓干细胞的增殖无明显影响,而能够显著提高ALP的活性,并呈现一定的剂量依赖性。经EMPs诱导后,细胞相对高表达DSPP、DMP-1mRNA。结论：EMPs对于SHED向成牙本质样分化具有积极作用。     

外泌体对脂多糖诱导的牙髓干细胞成骨分化的作用及代谢机制

目的:探究外泌体对脂多糖(LPS)诱导的牙髓干细胞(DPSCs)成骨分化的影响.方法:收集健康成人和儿童的脱落乳牙牙髓干细胞(SHED),分离、培养DPSCs和SHED并用流式细胞仪鉴定.收集SHED上清的外泌体,使用透射电镜、Western blot和粒径分析进行鉴定;检测对照组(10 mg/L PBS)、LPS组(10 mg/L LPS)和LPS+外泌体组(10 mg/L LPS+10 mg/L外泌体)的DPSCs细胞外酸化率(ECAR)和耗氧率(OCR)水平;加入线粒体呼吸酶抑制剂抗霉素A后,实时定量RT?PCR和茜素红染色检测各组DPSCs的成骨分化能力.结果:成功获得DPSCs和SHED来源外泌体;LPS可以提高DPSCs的ECAR水平,使OCR/ECAR水平下降,而加入外泌体后,DPSCs的OCR水平升高,OCR/ECAR水平得到恢复;外泌体可以恢复LPS诱导的成骨分化下降,而加入抗霉素A可以抑制外泌体的促成骨分化能力.结论:SHED来源的外泌体通过提高OCR/ECAR水平,来促进炎症微环境下DPSCs的成骨分化能力.     

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。     

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.     

HEMA对人乳牙牙髓干细胞分化潜能的影响

目的:探究HEMA对人乳牙牙髓干细胞向成牙本质细胞分化的影响。方法:将不同浓度的HEMA加入到SHED细胞中分别培养24 h、48 h和72 h后，MTT实验检测其毒性作用。将50 mg/mL抗坏血酸，10 mmol/L β-甘油磷酸钠和100 nmol/L地塞米松加入含10%胎牛血清的DMEM中制备矿化诱导液，无毒浓度HEMA(0.1 mmol/L和0.25 mmol/L)下，诱导SHED向成牙本质细胞分化，素红染色观察矿化结节的形成。结果:当HEMA浓度超过0.5 mmol/L,培养时间超过48 h时，HEMA对SHED细胞有毒性作用。长时间暴露于无毒浓度HEMA中，SHED细胞的分化潜能受到抑制。结论:HEMA可抑制SHED细胞向成牙本质细胞分化。     

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.     

SHED repair critical-size calvarial defects in mice

Objective:  Stem cells from human exfoliated deciduous teeth (SHED) are a population of highly proliferative postnatal stem cells capable of differentiating into odontoblasts, adipocytes, neural cells, and osteo-inductive cells. To examine whether SHED-mediated bone regeneration can be utilized for therapeutic purposes, we used SHED to repair critical-size calvarial defects in immunocompromised mice.
Materials and methods:  We generated calvarial defects and transplanted SHED with hydroxyapatite/tricalcium phosphate as a carrier into the defect areas.
Results:  SHED were able to repair the defects with substantial bone formation. Interestingly, SHED-mediated osteogenesis failed to recruit hematopoietic marrow elements that are commonly seen in bone marrow mesenchymal stem cell-generated bone. Furthermore, SHED were found to co-express mesenchymal stem cell marker, CC9/MUC18/CD146, with an array of growth factor receptors such as transforming growth factor β receptor I and II, fibroblast growth factor receptor I and III, and vascular endothelial growth factor receptor I, implying their comprehensive differentiation potential.
Conclusions:  Our data indicate that SHED, derived from neural crest cells, may select unique mechanisms to exert osteogenesis. SHED might be a suitable resource for orofacial bone regeneration.     

Comparison of human dental follicle cells (DFCs) and stem cells from human exfoliated deciduous teeth (SHED) after neural differentiation in vitro

Dental stem cells from human exfoliated deciduous teeth (SHED) and dental follicle cells (DFCs) are neural crest-derived stem cells from human dental tissues. Interestingly, SHED and DFCs can successfully differentiate into neuron-like cells. We hypothesized that SHED and DFCs have the same neural cell differentiation potentials. To evaluate neural cell differentiation, we cultivated SHED and DFCs in four different serum-replacement media (SRMs) and analyzed cell morphology, cell proliferation, and gene expression patterns before and after differentiation. In a standard cell culture medium, SHED and DFCs have not only similar cell morphologies, but they also have similar gene expression patterns for known stem cell markers. However, only SHED expressed the neural stem cell marker Pax6. After cultivation in SRMs, cell proliferations of DFCs and SHED were reduced and the cell morphology was spindle-like with long processes. However, differentiated DFCs and SHED had different neural cell marker expression patterns. For example, gene expression of the late neural cell marker microtubule-associated protein 2 was upregulated in DFCs and downregulated in SHED in SRM with the B27 supplement. In contrast, SHED formed neurosphere-like cell clusters in SRM with the B27 supplement, epidermal growth factor, and fibroblast growth factor-2. Moreover, SHED differentially expressed the glial cell marker glial fibrillary acidic protein, which in contrast was weakly or not expressed in DFCs. In conclusion, SHED and DFCs have different neural differentiation potentials under the same cell culture conditions.     

牙源性间充质干细胞诱导iPS细胞的效率与时间的研究

目的研究比较不同种牙源性间充质干细胞诱导iPS细胞的效率与时间。方法分离牙髓干细胞（DPSCs）、脱落乳牙干细胞（SHED）、牙乳头干细胞（SCAP）。应用慢病毒介导Lin28、Nanog、Oct4和Sox2因子重编程获得iPS细胞。比较在同等条件下三种细胞获得iPS细胞的克隆数与平均诱导时间。结果DPSC诱导iPS细胞的效率是0.167%,高于SHED细胞和SCAP细胞的诱导效率（0.125%,0.033%）;DPSC诱导iPS细胞的平均重编程时间是20.1d,均少于SHED细胞和SCAP细胞（23.73d,25.25d）,差异均有统计学意义。结论三种不同牙源性细胞有不同的iPS细胞诱导效率与重编程时间,牙髓干细胞有较好的诱导iPS细胞的应用潜能。     

Pulp tissue from primary teeth: new source of stem cells

SHED (stem cells from human exfoliated deciduous teeth) represent a population of postnatal stem cells capable of extensive proliferation and multipotential differentiation. Primary teeth may be an ideal source of postnatal stem cells to regenerate tooth structures and bone, and possibly to treat neural tissue injury or degenerative diseases. SHED are highly proliferative cells derived from an accessible tissue source, and therefore hold potential for providing enough cells for clinical applications. In this review, we describe the current knowledge about dental pulp stem cells and discuss tissue engineering approaches that use SHED to replace irreversibly inflamed or necrotic pulps with a healthy and functionally competent tissue that is capable of forming new dentin.     

脱落乳牙牙髓干细胞在口腔颌面部组织再生及免疫调节中应用的研究进展

乳牙牙髓干细胞是一种成体干细胞,提取自脱落的乳牙,易获取、无创伤,符合伦理要求,同时具有强增殖能力及多分化潜能;其低免疫原性的特点使得移植后宿主无发生免疫反应或排斥反应的风险,也不需要接受免疫抑制治疗.这些特性使乳牙牙髓干细胞成为再生医学中细胞治疗所需的理想种子细胞之一.文章就脱落乳牙牙髓干细胞(stem cells ...     

Dental pulp tissue engineering with stem cells from exfoliated deciduous teeth

Stem cells from human exfoliated deciduous teeth (SHED) have been isolated and characterized as multipotent cells. However, it is not known whether SHED can generate a dental pulp-like tissue in vivo. The purpose of this study was to evaluate morphologic characteristics of the tissue formed when SHED seeded in biodegradable scaffolds prepared within human tooth slices are transplanted into immunodeficient mice. We observed that the resulting tissue presented architecture and cellularity that closely resemble those of a physiologic dental pulp. Ultrastructural analysis with transmission electron microscopy and immunohistochemistry for dentin sialoprotein suggested that SHED differentiated into odontoblast-like cells in vivo. Notably, SHED also differentiated into endothelial-like cells, as demonstrated by B-galactosidase staining of cells lining the walls of blood-containing vessels in tissues engineered with SHED stably transduced with LacZ. This work suggests that exfoliated deciduous teeth constitute a viable source of stem cells for dental pulp tissue engineering.     

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。