牙周膜干细胞成脂分化的研究进展

牙周膜干细胞( periodontal ligament stem cells,PDLSCs)被认为是牙周组织工程理想的种子细胞,具有多向分化潜能。间充质干细胞在成脂和成骨分化过程中具有相互制约的平衡关系,成脂分化的诱导因素通常是成骨分化的抑制因素,反之亦然。本文就近年来PDLSCs成脂分化的诱导、鉴定方法及相关转录因子的研究进展进行综述,分析PDLSCs成脂分化的机制,探讨通过抑制PDLSCs成脂分化促进其成骨分化的可行性,为治疗牙槽骨吸收等骨骼性疾病提供参考。     

TNF-α对三种牙源性干细胞增殖分化能力影响的比较研究

目的 探讨同一供体来源的人牙龈干细胞（human gingival mesenchymal stem cells, hGMSCs）,人牙髓干细胞（human dental pulp stem cells, hDPSCs）及人牙周膜干细胞（human periodontal ligament stem cells, hPDLSCs）的增殖、成骨成脂分化能力及特定浓度的肿瘤坏死因子-α(tumour necrosis factor-α, TNF-α)对三者性能的影响,进而为牙周组织再生过程中种子细胞的选取及优化提供策略。方法 分离培养并鉴定hGMSCs、hDPSCs和hPDLSCs。将第3代细胞分别于正常和含10 ng/mL TNF-α的成骨、成脂诱导液中连续培养21天,7天时检测ALP(alkaline phosphatase,ALP)水平,21天时茜素红染色测定成骨能力,油红O染色测定成脂能力。结果 三种干细胞中h GMSCs的增殖及成脂能力最强（P<0.05）,h PDLSCs的成骨能力最强（P<0.05）,hDPSCs在增殖及分化方面表现居中。10 ng/mL TNF-...     

年龄因素对人牙周膜干细胞培养和性能的影响

目的:探讨年龄因素对人牙周膜干细胞(periodontal ligament stem cells,PDLSCs)体外培养及其生物学特性的影响。方法:收集临床<30岁、>50岁具有完整牙根的第三磨牙各4个,分别刮取根面残留牙周膜进行PDLSCs分离培养,观察其细胞克隆形成能力、MTT方法检测生长曲线、流式细胞仪分析细胞表面分子,并进行体外成骨、成脂诱导,对比分析PDLSCs多向分化能力。结果:两组第三磨牙中均可培养出具有明显间充质干细胞(mesenchymal stem cell,MSC)特性的PDLSCs,但>50岁组获得的PDLSCs克隆形成率显著降低(P<0.05),其增殖、分化能力亦较<30岁组明显减弱(P<0.05)。结论:年龄因素对PDLSCs体外培养及其生物学特性具有明显影响;PDLSCs的基础和临床研究中,应充分考虑到病人年龄因素。     

人牙周膜干细胞的初步鉴定及体外成骨诱导

目的：体外培养、鉴定人牙周膜干细胞（periodontal ligament stem cells,PDLSCs）并定向诱导分化为成骨细胞,探讨人PDLSCs的多向分化潜能：方法：体外分离、培养人牙周膜细胞,待细胞达一定量后用有限稀释法进行克隆化培养,筛选鉴定牙周膜干细胞（PDLSC）,矿化诱导培养21d后检测钙结节形成情况、ALP活性,免疫细胞化学检测骨涎蛋白（BSP）、Ⅰ型胶原表达,RT—PCR检测ALP、BSP mRNA表达。结果：人PDLSCs体外诱导培养21d后可见钙结节形成,成骨细胞相关蛋白及mRNA均阳性表达。结论：人PDLSCs在体外诱导条件下具有成骨潜能。     

牙髓干细胞的研究与应用

牙髓干细胞（dental pulp stem cells,DPSCs）是牙髓组织中高度增殖、具有自我更新能力和多向分化潜能的一类成体干细胞,在牙齿、骨和神经的修复、再生以及组织工程相关研究中具有重要作用。本文主要从 DPSCs 的生物学特性、分化潜能以及组织工程应用等方面综合分析了 DPSCs 研究的进展。     

大鼠骨髓间充质干细胞与脂肪间充质干细胞生物学特性的比较

目的:本研究旨在比较同一个体来源的脂肪间充质干细胞(Adipose tissue-derived mesenchymal stem cells,ADSCs)和骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)的生长特点和诱导分化能力。方法:取7～10 d SD大鼠的脂肪和骨髓,体外分离、纯化脂肪和骨髓来源间充质干细胞,分别从细胞形态、生长动力学、表面标志、分化潜能等方面进行鉴定和比较。结果:脂肪间充质干细胞的增殖速度明显大于骨髓间充质干细胞(P<0.05),两种细胞所表达的表面蛋白标志物基本相似,但脂肪间充质干细胞表面蛋白标志CD106呈阴性,而骨髓间充质干细胞CD106呈阳性。在成软骨分化中脂肪间充质干细胞弱表达Ⅱ型胶原,而骨髓间充质干细胞不表达。第5、10、15、20代ADSCs及BMSCs经成骨诱导后茜素红染色均呈阳性且有"矿化"结节形成,碱性磷酸酶活性以第5代最强,随着细胞代次的递增,碱性磷酸酶活性呈递减趋势。结论:ADSCs较BMSCs更易于分离培养及体外扩增,诱导条件下成骨、成脂、成软骨能力较强,适合作为再生医学的种子细胞。     

大气压常温等离子体对人牙周膜干细胞成骨分化的影响

目的:探究大气压常温等离子体(atmospheric room temperature plasma, ARTP)对人牙周膜干细胞(human periodontal ligament stem cells, hPDLSCs)成骨分化的影响。方法:原代培养人牙周膜细胞,采用免疫磁珠法从中分选出hPDLSCs,通过成骨和成脂诱导培养检测其分化潜能;利用不同时长ARTP处理hPDLSCs,行成骨诱导培养,测定细胞碱性磷酸酶(alkaline phosphatase, ALP)活性,茜素红染色(alizarin red staining, ARS)及半定量分析法观察细胞矿化结节形成状况,RT-qPCR测定细胞成骨相关基因的表达状况,测定细胞内活性氧粒子(reactive oxygen species, ROS)含量变化。结果:采用免疫磁珠法分选出的hPDLSCs呈长梭形、多角形,表现出良好的成骨和成脂分化潜能;ARTP处理时长1 min可提高hPDLSCs的ALP活性,增加细胞内矿化结节形成量;能够提升hPDLSCs成骨相关基因ALP、COL-I、RUNX2的表达水平;ARTP提高了hPDL...     

化脓链球菌脂磷壁酸对人牙周膜干细胞分化能力的影响

目的 研究脂磷壁酸(LTA)对牙周膜干细胞(PDLSCs) 分化能力的影响,从而探讨G+细菌毒素LTA对人牙周膜干细胞再生牙周组织的影响.方法 用0.1、1和10μg/ml的LTA作用于PDLSCs,采用MTT方法及流式细胞术检测细胞的增殖及凋亡情况,使用碱性磷酸酶染色和茜素红染色对细胞的成骨分化能力进行比较;应用油红O染色鉴定成脂分化能力,实时定量PCR检测成骨成脂相关基因的变化,研究LTA在PDLSCs分化中的作用.结果 1μg/ml及10μg/ml LTA显著降低PDLSCs增殖率,细胞凋亡增加.ALP染色及活性结果表明,早期成骨能力各组之间没有显著差异;茜素红染色结果及钙离子浓度定量测定显示,随LTA浓度升高,PDLSCs晚期成骨能力降低,并具有LTA浓度依赖性.成骨相关基因Runx2表达表现相同变化趋势.油红O染色结果表明,随着LTA浓度的增加,PDLSCs的成脂分化能力升高,成脂相关基因PPARγ表达升高,具有LTA浓度依赖性.结论 LTA降低PDLSCs成骨分化能力,同时促进细胞成脂分化能力,表明LTA抑制PDLSCs介导的牙周组织再生.     

人脐带Wharton's Jelly来源间充质干细胞与人牙周膜干细胞成骨分化能力的对比研究

目的:比较人脐带Wharton's Jelly来源间充质干细胞(human umbilical cord Wharton's Jelly-derived mesechymal stem ceils,hUCWJMSCs)与人牙周膜干细胞(periodontal mesenchymal stem cells,hPDLSCs)成骨分化能力.方法:体外培养hUC-WJMSCs和hPDLSCs.MTT法检测细胞增殖情况;成骨诱导后测定细胞的ALP活性,茜素红染色检测细胞矿化能力,Real-timePCR分析OPN和Runx2基因的表达.结果:hUCWJMSCs增殖能力高于hPDLSCs;经矿化诱导后hPDLSCs ALP表达、矿化结节形成高于hUCWJMSCs(P＜0.05);Runx2在hPDLSCs中表达高于hUCWJMSCs(P ＜0.05);而hUCWJMSCs中OPN表达高于hPDLSCs(P＜0.05).结论:hUCWJMSCs、hPDLSCs均具有成骨分化能力,hPDLSCs成骨分化能力较强.     

Mesenchymal stem cells derived from dental tissues

Regeneration of tissues occurs naturally due to the existence of stem cells with the capacity to self-regenerate and differentiate; however, regenerative capacity decreases with age, and in many cases, regeneration is not sufficient to repair the damage produced by degenerative, ischaemic, inflammatory, or tumour-based diseases. In the last decade, advances have been made in the understanding of stem cells, the genes that control the alternative fates of quiescence and differentiation, and the niches that provide specific signals that modulate cell fate decisions. Embryonic stem-cell research is shedding light on the secrets of development. Adult stem cells (AS cells) are available from several sources. Bone marrow and connective tissue have been used in preliminary clinical trials for regenerative therapy. Recently, several types of AS cells have been isolated from teeth, including dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, dental follicle progenitor stem cells and stem cells from apical papilla. Preliminary data suggest that these cells have the capacity to differentiate into osteoblasts, adipocytes, chondrocytes and neural cells. If confirmed, these data would support the use of these cells, which are easily obtained from extracted teeth, in dental therapies, including in regenerative endodontics, providing a new therapeutic modality.     

人源类器官的研究进展及在口腔医学的展望

类器官是指在体外三维培养构建的,依赖于人造细胞外基质的多细胞团,具有自我更新、自我组织能力,并维持了其来源组织的生理结构和功能。类器官作为一种新兴的研究模型,兼具细胞系和动物模型两者的优点。其构建材料简单、培养效率高、耗时短,在疾病研究模型构建、临床药敏试验和再生医学中有良好的应用前景。类器官模型主要分为三种细胞来源类型：胚胎干细胞/诱导多能干细胞、成体干细胞、肿瘤细胞。本文将重点介绍三种细胞来源类器官最新的研究进展,并展望其在口腔医学中的应用。     

Immunomodulatory properties of dental tissue‐derived mesenchymal stem cells

In addition to their well‐established self‐renewal and multipotent differentiation properties, mesenchymal stem cells (MSCs) also possess potent immunomodulatory functions both in vitro and in vivo, which render them a potential novel immunotherapeutic tool for a variety of autoimmune and inflammation‐related diseases. The major mechanisms may involve (1) the secretion of an array of soluble factors such as prostaglandin E₂ (PGE₂), indoleamine 2, 3‐dioxygenase (IDO), transforming growth factor‐β (TGF‐β), and human leukocyte antigen G5 (HLA‐G5); (2) interactions between MSCs and immune cells such as T cells, B cells, macrophages, and dendritic cells. Recently, increasing evidence has supported that MSCs derived from dental tissues are promising alternative sources of multipotent MSCs. We here provide a thorough and extensive review about new findings in the immunomodulatory functions of MSCs derived from several dental tissues, including dental pulp, periodontal ligament, gingiva, exfoliated deciduous teeth, apical papilla, and dental follicle, respectively. The immunomodulatory properties of dental MSCs place them as a more accessible cell source than bone marrow‐derived MSCs for cell‐based therapy of immune and inflammation‐related diseases.     

Oct4调节成体干细胞多能性的研究进展

成体干细胞是存在于人和哺乳动物特定组织中的具有自我更新和一定分化潜能的未成熟细胞,参与成体组织的更新和创伤修复.牙髓干细胞是牙髓损伤修复的潜能所在,如何调控其生物学行为以促进牙髓修复再生是当前的研究热点.Oct4 在干细胞发育的基因调控网络中居核心地位,近年研究显示Oct4 具有维持成体干细胞多能性的功能.本文通过阐述Oct4 的结构和功能,揭示其在成体干细胞中的调节作用,为研究Oct4 对牙髓干细胞的调节作用提供理论基础.     

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。     

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.     

颌突外胚间充质干细胞参与颅骨缺损修复的作用

目的：探讨颌突外胚间充质干细胞参与颅骨缺损修复的作用。方法：将外胚间充质干细胞复合煅烧骨后移植入大鼠颅骨缺损处,以大鼠成纤维细胞、空白胶原作为对照,1、3、6个月后取材,经HE染色,Mallory改良三色法鉴定。结果：HE染色结果显示实验组成骨的效果明显好于对照组;图像分析见实验组的骨基质面积明显多于对照组,统计学分析P〈0.01,有明显差异。结论：提示移植的外胚间充质干细胞在局部颅骨组织内存活并具有一定的功能。     

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。