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.     

共培养诱导人脐带间充质干细胞(hUCMSCs)向牙周膜细胞(hPDLs)分化的体外实验研究

目的:研究人脐带间充质干细胞(hUCMSCs)向人牙周膜细胞(hPDLs)分化的可能性。方法:原代培养获得hUCMSCs和hPDLs;应用Transwell小室将两种细胞进行非接触式共培养;使用流式细胞仪检测共培养过程中hUCMSCs干细胞标记物CD146、SOX2、SSEA和Stro-1的变化情况;通过免疫荧光技术和Western Blot技术检测共培养过程中波形蛋白(Vimentin)在hUCMSCs中的变化情况。结果:共培养过程中hUCMSCs中的CD146、SOX2、SSEA和Stro-1持续降低;而Vimentin持续增高。结论:通过与hPDLs共培养,可以成功地将hUCMSCs诱导分化成为牙周膜样细胞,进一步证明了hUCMSCs可用于牙周损伤修复的可能。     

人牙周膜干细胞的分离培养及体外诱导分化研究

目的:从成体人牙周组织中分离培养牙周膜干细胞,研究其生物学特性并进行诱导分化,为牙周组织工程提供可靠的种子细胞来源.方法:选取12～20岁的年轻患者因正畸拔除的健康牙齿,采用酶消化组织块培养法得到牙周膜细胞,待细胞达一定量后用有限元稀释法进行单细胞克隆,筛选牙周膜干细胞( PDLSCs).计算细胞克隆形成率(CFU- ...     

牙髓干细胞、外胚间充质干细胞裸鼠体内分化的实验研究

目的：将免疫磁珠STRO＋-1的牙髓干细胞（DPSC）和外胚间充质干细胞（EMSC）与支架材料复合,观察在裸鼠体内移植后的分化能力,为干细胞分化能力研究提供依据.方法：收集免疫磁珠STRO＋-1的DPSC和EMSC,将其与陶瓷化骨复合移植入裸鼠背部皮下,8周后移植物组织学切片,HE染色观察及免疫组织化学检测DSP分布.结果：DPSC组移植物中可见新生基质,其相邻的结缔组织细胞部分出现极化,似成牙本质样细胞,局部放大可见类似于不典型的牙本质牙髓复合体.EMSC组有基质形成,结构不典型,有骨样基质形成,也存在有类牙本质牙髓复合体样结构.在移植后8周,DPSC组DSP在沉积的新生基质和部分出现极化的细胞胞浆中呈阳性表达.结论：通过免疫磁珠筛选的STRO＋-1的DPSC具有自我更新的特性,能够在体内继续分化形成牙本质样基质.EMSC组移植到裸鼠皮下,也能够形成基质.但是结构不典型.     

人脐带间充质干细胞与牙髓细胞体外共培养对细胞生物学的影响

目的: 研究人脐带间充质干细胞（human umbilical cord mesenchymal stem cells, hUCMSCs）与经骨形态发生蛋白2（bone morphogenetic protein 2,BMP2）诱导后的人牙髓细胞（human dental pulp cells,hDPCs）共培养对细胞生物学特性的影响。方法: 分别取原代培养的hUCMSCs和hDPCs,分别通过流式细胞术、成骨诱导、成脂诱导以及免疫组织化学染色鉴定细胞;使用BMP2诱导hDPCs,14 d后检查其DSPP、ALP、DMP1的mRNA表达情况;按照1∶1、1∶5、5∶1的比例直接共培养hUCMSCs与经BMP2诱导后的hDPCs,实时定量PCR检测其DSPP、ALP、DMP1、OCN、VEGF、HGF、Nanog的mRNA表达情况。根据实时定量PCR结果选取1∶1组与单独培养hUCMSCs组、hDPCs组比较,分别培养21 d后进行茜素红染色,在酶标仪上于562 nm处检测沉淀物形成情况。采用SPSS21.0软件包对数据进行统计学分析。结果: 直接共培养14 d后,1∶1细胞组的DSPP、ALP、DMP1、OCN、VEGF、HGF的mRNA表达量比单独培养的hUCMSCs组显著升高（P<0.05）,而Nanog mRNA表达量比单独培养的hUCMSCs组降低（P<0.05）。茜素红染色结果显示,1∶1组的OD值显著高于hUCMSCs组（P<0.05）。结论: 将hUCMSCs与经BMP2诱导后的hDPCs按照1∶1比例共培养,可以诱导细胞向成牙本质细胞方向分化,并促进血管生成因子的表达。     

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。     

人牙髓干细胞体外分选分化的初步实验研究

目的体外培养人牙髓细胞,分选牙髓干细胞并诱导其分化。方法选择因正畸目的而拔除的健康完整双尖牙,酶消化法进行牙髓细胞培养,并进行来源鉴定。单抗Stro-1标记牙髓干细胞、免疫磁珠分选系统进行分选。矿化液定向诱导分选后的牙髓干细胞,比较诱导前后stro-1染色及改良Gomori钙钴法染色检测碱性磷酸酶（ALP）的改变。结果体外培养人牙髓细胞呈成纤维细胞样,抗波形丝蛋白染色阳性,抗角蛋白染色阴性。牙髓干细胞Stro-1检测阳性,牙髓细胞中干细胞阳性率约为10％。矿化诱导后细胞stro-1阴性、ALP阳性表达。结论采用免疫磁珠分选系统分离出人牙髓干细胞,初步验证干细胞分化潜能,为其后续生物学特性研究提供实验基础。     

人牙周韧带细胞和牙髓细胞表型特征的比较

目的研究人牙周韧带细胞（PDLCs）和牙髓细胞（DPCs）表型特征以及体外传代对其表型特征的影响,为选择适宜的表面标记分选生物性状同源性的细胞亚群提供依据。方法采用酶消化法体外培养PDLCs和DPCs,免疫组化检测和流式细胞仪分析细胞表面标记的表达。结果PDLCs和DPCs的STRO-1和CD146免疫组化染色阳性。流式细胞仪分析显示：获得的第1代PDLCs和DPCs表达间充质干细胞表面标记STRO-1、CD146、CD29、CD44和CD106,基本不表达CD34。PDLCs的STRO-1、CD29和CD44阳性表达百分比与DPCs间的差异无统计学意义（P>0.05）,PDLCs的CD106阳性表达百分比高于DPCs,CD146阳性表达百分比低于DPCs,且差异有统计学意义（P<0.001）。PDLCs和DPCs表达STRO-1和CD146的阳性表达百分比随传代而逐渐减低。结论PDLCs表面抗原表达与DPCs相似,而且其中成体干细胞的成分随传代逐渐减少。     

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.     

Effect of leptin on differentiation of human dental stem cells

Oral Diseases (2011) 17 , 662–669 Objectives: Mesenchymal stem cells (MSCs) were identified in adult human periodontal ligament and dental pulp that are considered as potential stem cell sources for future clinical applications in dentistry. Leptin is known as an important regulator of mesenchymal differentiation. The objective of this study was to elucidate the role of leptin on proliferation and differentiation of dental MSCs. Materials and methods: Enhancement of cemento/odontoblastic differentiation of dental stem cells by leptin was confirmed by alizarin red S staining and alkaline phosphatase activity staining. In contrast, leptin reduced adipogenesis in both dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) confirmed by oil red O staining and RT‐PCR. The expression of adipogenic markers, lipoprotein lipase and proliferator‐activated receptor γ2 (PPARγ2), were suppressed in PDLSCs incubated on media supplemented with leptin for 2 weeks. Results: Leptin had a relatively stronger osteogenesis promoting effect and adipogenesis suppressing effect in PDLSCs than in DPSCs. Conclusions: Collectively, leptin had a relatively stronger promoting effect on cemento/odontoblastic differentiation and a suppressing effect on adipogenesis in PDLSCs than in DPSCs. This study has provided evidence that leptin acts as an important modulator of dental MSCs differentiation.     

组蛋白乙酰化在慢性牙周炎来源牙周膜干细胞成骨分化中的作用

表观遗传是指基因序列不发生改变的前提下,基因表达却发生改变,且这种改变能够遗传至后代。其中,组蛋白乙酰化属于表观遗传范畴中重要的一种类型。现有的研究表明慢性牙周炎的发生与表观遗传修饰具有一定的关系。结合已有研究,本文对组蛋白乙酰化在慢性牙周炎来源牙周膜干细胞成骨分化中的作用作一综述。     

牙髓干细胞分化的研究进展

牙髓干细胞(DPSC)是一种具有高度增生、自我更新能力和多相分化潜能的成体干细胞,在一定条件下可向特定的细胞类型分化,在牙髓修复和牙齿再生中发挥着重要的作用。本文主要就DPSC成骨向分化、成牙本质向分化的研究进展作一综述。     

The efficacy of mesenchymal stem cells to regenerate and repair dental structures

OBJECTIVES: Identification, characterization, and potential application of mesenchymal stem cells (MSC) derived from human dental tissues. METHODS: Dental pulp and periodontal ligament were obtained from normal human impacted third molars. The tissues were digested in collagenase/dispase to generate single cell suspensions. Cells were cultured in alpha-MEM supplemented with 20% fetal bovine serum, 2 mM l-glutamine, 100 microM l-ascorbate-2-phosphate. Magnetic and fluorescence activated cell sorting were employed to characterize the phenotype of freshly isolated and ex vivo expanded cell populations. The developmental potential of cultured cells was assessed following co-transplantation with hydroxyapetite/tricalcium phosphate (HA/TCP) particles into immunocompromised mice for 8 weeks. RESULTS: MSC were identified in adult human dental pulp (dental pulp stem cells, DPSC), human primary teeth (stem cells from human exfoliated deciduous teeth, SHED), and periodontal ligament (periodontal ligament stem cells, PDLSC) by their capacity to generate clongenic cell clusters in culture. Ex vivo expanded DPSC, SHED, and PDLSC populations expressed a heterogeneous assortment of makers associated with MSC, dentin, bone, smooth muscle, neural tissue, and endothelium. PDLSC were also found to express the tendon specific marker, Scleraxis. Xenogeneic transplants containing HA/TCP with either DPSC or SHED generated donor-derived dentin-pulp-like tissues with distinct odontoblast layers lining the mineralized dentin-matrix. In parallel studies, PDLSC generated cementum-like structures associated with PDL-like connective tissue when transplanted with HA/TCP into immunocompromised mice. CONCLUSION: Collectively, these data revealed the presence of distinct MSC populations associated with dental structures with the potential of stem cells to regenerate living human dental tissues in vivo.