乳牙牙髓干细胞体外诱导成骨细胞的实验研究

目的:体外分离、培养人乳牙牙髓干细胞,并向成骨细胞诱导.方法:采用酶消化法分离获得人乳牙牙髓干细胞,有限稀释法分离纯化,测定细胞克隆形成率,细胞计数法测定生长曲线,细胞爬片行HE染色,抗波形蛋白(Vimentin)、CD44和STRO-1免疫组化染色,并向成骨细胞诱导,行HE染色、碱性磷酸酶染色、Von Kossa染色、Van Gieson染色和抗骨钙素免疫组化染色进行鉴定.结果:通过有限稀释法获得了人乳牙牙髓干细胞,并诱导成成骨细胞,表现出与典型成骨细胞相似的形态特征和生物学特征.结论:成功从人乳牙牙髓中分离出牙髓干细胞,并诱导为成骨细胞.     

山羊乳牙牙髓细胞体外培养和绿色荧光转染的研究

目的:体外分离、培养山羊乳牙牙髓细胞,并转染绿色荧光蛋白.方法:采用组织块法获得山羊乳牙牙髓细胞,细胞计数法测定生长曲线.细胞爬片行HE染色、碱性磷酸酶染色、抗波形蛋白和角蛋白免疫组化染色.加入含GFP反转录病毒液转染山羊乳牙牙髓细胞.结果:通过组织块法获得了山羊乳牙牙髓细胞,细胞群体倍增时间为43.79h.HE染色显示细胞形态为梭形,细胞体小,胞核大.细胞碱性磷酸酶染色阳性,抗波形蛋白免疫组化染色阳性,抗角蛋白免疫组化染色阴性.转染绿色荧光蛋白后,细胞能持续表达绿色荧光.结论:山羊乳牙牙髓组织可分离培养出间叶来源的乳牙牙髓细胞,并成功使之表达绿色荧光.     

人牙周膜干细胞的分离培养及初步鉴定

目的 体处分离培养人类牙周膜干细胞并对其生物学性状进行初步鉴定.方法 采用有限稀释法进行牙周膜干细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、免疫荧光染色等方法鉴定牙周膜干细胞的组织来源及生物学特性.结果 有限稀释法获得的牙周膜干细胞克隆株呈簇状生长,排列紧密,细胞呈圆形或椭圆形,细胞较小,排列形成紧密的团块状.免疫组化染色显示细胞波形蛋白阳性,角蛋白阴性;碱性磷酸酶染色阳性;早期间充质干细胞的标志物STRO-1荧光染色显示细胞发出明亮的红色荧光,表达阳性.结论 牙周膜中存在具有高增殖能力的干细胞,克隆化分离培养的人牙周膜干细胞具有强克隆形成能力,具有间充质干细胞表型和生物学特性.     

犬乳牙牙髓干细胞体外培养及生物学性能研究

目的：体外分离培养比格犬乳牙牙髓干细胞（DTPSCs）,研究其生物学特性。方法：用酶解组织块法培养6周龄比格犬 DTPSCs,克隆法纯化,检测细胞克隆形成能力及增殖曲线;免疫组化鉴定细胞来源,流式细胞术鉴定细胞表面标记物;进行细胞多向诱导分化能力检测。结果：获得了成集落状生长的犬乳牙牙髓细胞,其克隆形成率为32％;免疫组化确定细胞为间充质而非造血来源;流式细胞术鉴定 STRO-1、CD146阳性表达,而 CD14、CD45及 CD86阴性表达;细胞成骨、成脂诱导后分别对茜素红、油红 O 染色阳性,成牙本质诱导后细胞碱性磷酸酶、牙本质涎磷蛋白染色阳性,且细胞内碱性磷酸酶活性显著增高。结论：比格犬乳牙牙髓中存在具有间充质干细胞表型、自我更新及多向分化能力的干细胞。     

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。     

人牙髓干细胞的体外分离、培养及鉴定

目的 :体外分离、培养人牙髓干细胞 ,从不同角度对其进行鉴定。方法 :采用胶原酶和Dispase酶联合消化法培养人牙髓干细胞 ,有限稀释法分离纯化 ,将克隆形成的细胞通过透射电镜观察其超微结构、流式细胞仪细胞周期分析及抗波形丝蛋白 (Vimentin)、CD44、骨粘素 (ON)、牙本质涎蛋白 (DSP)免疫组化染色方法进行鉴定。结果 :通过有限稀释法获得了人牙髓干细胞克隆 ,透射电镜下观察这种克隆形成细胞具有干细胞典型的超微结构特点 ,大多数细胞处于G0 /G1期 ,为细胞周期的静止期 ,并具有牙源性未分化间充质细胞的表型特点。结论 :克隆化培养是人牙髓干细胞较为有效的分离纯化方法 ,该方法分离的人牙髓干细胞具有干细胞的结构、细胞周期及表型特点。     

EMPs对人牙髓干细胞增殖分化影响的实验研究

目的：将人牙髓干细胞（human Dental Pulp Stem Cells,hDPSCs）与釉基质蛋白（Enamel Matrix Proteins,EMPs）按一定浓度结合,测定牙本质涎磷蛋白（dentin sialoprotein,DSPP）、波形蛋白（vimentin）、碱性磷酸酶（alkaline phophatase,ALP）的表达,阐述EMPs对人牙髓干细胞增殖分化的影响.方法：分别将浓度为100 μg/mL、200μg/mL的EMPs加入培养中的人牙髓干细胞中,分别于3d、5d、7d、9d、11d对细胞进行免疫组化染色,检测细胞爬片DSPP、vimentin、ALP的表达.结果：未经EMPs诱导的人牙髓干细胞DSPP少量细胞阳性表达,vimentin表达阴性,ALP表现低活性.经200 μg/mL的EMPs诱导5d后,人牙髓干细胞DSPP、vimentin染色呈阳性表达,ALP活性明显增加.结论：EMPs对人牙髓干细胞增殖及分化具有促进作用,200 μg/mL的浓度效果最为显著.     

犬牙周膜干细胞体外分离培养和鉴定的实验研究

目的体外分离培养犬牙周膜干细胞,并对其进行生物学鉴定。方法采用有限稀释法进行犬牙周膜细胞克隆筛选,获得单细胞克隆来源细胞,检测其克隆形成率,并采用免疫组织化学染色、碱性磷酸酶染色、苏木精-伊红染色以及生长因子分化诱导等方法观察其生物学特性。结果1）克隆化分离培养的犬牙周膜干细胞呈集落状生长,增殖快,克隆形成率为0.95％。2）苏木精-伊红染色见细胞呈成纤维样,体积小、核大,有较多的双核细胞。3）碱性磷酸酶染色阳性。4）细胞表型组化鉴定波形丝蛋白、Ⅰ型胶原、Ⅲ型胶原阳性,间充质干细胞表面标志STRO-1染色阳性,角蛋白染色阴性。5）在含β-磷酸甘油钠、维生素C、地塞米松的矿化液中,细胞被诱导分化为成骨细胞,碱性磷酸酶活性增强,Ⅲ型胶原染色阴性,能形成矿化结节,细胞表达成骨细胞特异的骨涎蛋白,在含β-羟基乙醇的培养液中,被诱导分化为神经细胞样细胞。结论克隆化分离培养的犬牙周膜干细胞具有很强的克隆形成能力,具有间充质干细胞表型和多向分化潜能。     

成体人牙髓干细胞的分离与鉴定

目的从成体人牙髓组织中分离培养牙髓干细胞，初步探讨其分化潜能。方法选取年轻患者因正畸或阻生拔除的健康第三磨牙，取出牙髓，采用酶消化及过滤法得到单细胞悬液，有限稀释法原代培养。扩大培养细胞克隆，检测STRO-1的表达。体外诱导分化后对各克隆从碱性磷酸酶(ALP)活性、矿化结节形成、牙本质涎蛋白(DSP)表达、Oil Red—O染色、PPARr2基因表达等方面进行检测。结果克隆来源细胞STRO-1表达阳性。在矿化液诱导下，克隆细胞呈现明显高的ALP活性；能够形成矿化结节；可以分泌表达DSP，已向成牙本质细胞方向发生了分化。成脂肪诱导后，Oil Red—O染色阳性，可检测到PPARr2基因表达。结论从成体人牙髓组织中可以分离培养出干细胞，在体外能有效增殖并保持低分化状态。     

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十细胞,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,牙髓细胞中干细胞附性率约为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.     

CM-DiI体外标记人乳牙牙髓干细胞的可行性研究

目的: 筛选CM-DiI用于人乳牙牙髓干细胞标记的最佳浓度,并对标记后细胞的生物学行为进行评价,为下一步细胞活体移植和体内示踪打下基础。方法: 酶解组织块法培养人乳牙牙髓细胞并纯化,免疫组化鉴定细胞来源,进行细胞体外多向诱导分化能力实验。将第3代细胞分别使用浓度2 mg/L、3 mg/L及4 mg/L的CM-DiI进行标记,比较标记后细胞乳酸脱氢酶释放率及细胞增殖情况,并观察经体外多次传代后细胞荧光的表达情况。结果: 酶解组织块法可获得成集落状生长的人乳牙牙髓细胞;免疫组化确定细胞为间充质而非造血来源;细胞具有成脂、成骨及成牙本质等多向分化能力。3种标记物浓度均未对细胞乳酸脱氢酶释放及细胞增殖产生显著的不利影响;随体外多次传代,标记荧光强度逐渐减退。结论: CM-DiI操作简便、染色速度快,可有效的标记人乳牙牙髓干细胞,其中3 mg/L的标记浓度细胞毒性极小,标记效率高,在体外多次传代后荧光信号仍能稳定表达。     

Isolation and characterization of dental pulp stem cells from a supernumerary tooth

Background: Dental pulp stem cells (DPSCs) were primarily derived from the pulp tissues of primary incisors and permanent third molar teeth, whereas no report to our knowledge has yet been documented on deriving DPSCs from the other tooth types. The aim of this study is to present a novel approach of harvesting stem cells from a supernumerary tooth (a mesiodens). Materials and methods: The pulp tissues from a mesiodens of a 20‐year‐old healthy male patient and the left lower deciduous canine of a healthy 10‐year‐old boy (the positive control) were extracted and cultured for DPSCs, which were examined with stem cells (Oct‐4, Nanog and Rex‐1) and differentiation (Osteonectin and Nestin) markers. Furthermore, DPSCs were directionally differentiated to osteogenic and adipogenic cell lineages. Results: Dental pulp stem cells derived from the mesiodens were capable of differentiating into adipogenic and osteogenic lineages. The mesioden’s DPSCs also expressed stem cell and differentiation markers, which suggested their stem cell origin and differentiation capability. All the aforementioned results for the mesiodens were consistent with those of the DPSCs derived from the positive control. Conclusion: We have demonstrated the feasibility of deriving DPSCs from a usually discarded tissue such as a supernumerary tooth.     

Dental pulp cells derived from permanent teeth express higher levels of R-cadherin than do deciduous teeth: implications of the correlation between R-cadherin expression and restriction of multipotency in mesenchymal stem cells

ObjectivesThe aim of this study was to characterize the expression status of cadherins in dental pulp-derived mesenchymal progenitor/stem cells from deciduous and permanent teeth, and to determine how cadherins affect the multipotency of the progenitor/stem cells.Materials and methodsWe evaluated and compared the expression status of cadherins in dental pulp-derived cells from deciduous teeth and in cells from permanent teeth by using an array of primers for amplification of RNA encoding human cell adhesion molecules and a real time PCR system. In order to elucidate how cadherins (which are differentially expressed in deciduous and permanent teeth) affect the multipotency of the dental pulp-derived progenitor/stem cells, the ability of the dental pulp cells to differentiate into adipocytes and osteoblasts was evaluated.ResultsR-cadherin was found to be vigorously expressed in the dental pulp cells derived from permanent teeth but not in the dental pulp cells derived from deciduous teeth. N-cadherin was found to be expressed essentially equally in both types of cells. The ability of the dental pulp cells of deciduous teeth to differentiate into adipocytes and osteoblasts was found to be much higher than that of cells obtained from permanent teeth.ConclusionR-cadherin may be a key molecule for providing control over the multipotency of the dental pulp-derived mesenchymal stem cells.     

Cbfa1/Runt2在牙髓组织和牙髓干细胞中的表达研究

目的:检测Cbfa1/Runt2在成人牙髓组织及牙髓细胞的表达,以便进一步探讨其在成牙本质细胞分化过程中所起的作用.方法:选取因正畸拔除的第一前磨牙牙髓,一部分用于免疫酶标检测,一部分用酶消化法进行细胞培养.取第4代细胞进行Cbfa1/Runt2的免疫荧光检测.选取牙龈组织和牙龈成纤维细胞作为对照.结果:Cbfa1/Runt2在牙髓组织的表达集中在成牙本质细胞下层和血管周围.在牙髓细胞的表达集中在细胞核.结论:Cbfa1/Runt2可能参与成牙本质细胞分化的调控,但其在成牙本质细胞分化过程中所起的作用还需进一步研究.     

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.     

Proliferative activity of cells from remaining dental pulp in response to treatment with dental materials

Background:  The biological examination of pulp injury, repair events and response of dental pulp stem cells to dental restorative materials is important to accomplish restorative treatment, especially to commonly used dental materials in paediatric dentistry, such as glass ionomer cement (GIC) and calcium hydroxide (Ca(OH)₂) lining cement.
Methods:  Healthy patients aged between 9 to 11 years with carious primary molars without pulp exposure were selected and divided into two groups: Group 1 (teeth restored with GIC) and Group 2 (teeth lined using Ca(OH)₂ and restored with GIC). The proliferative activity of stem cells of teeth between these two groups was compared using colourimetric cell proliferation reagent, alamarBlue. Immunocytochemistry and flow cytometry confirmation were performed using mesenchymal stem cell markers, CD105 and CD166.
Results:  The proliferative activity using alamarBlue™ assay showed that cells derived from the remaining dental pulp of exfoliated deciduous teeth were positive for CD105 and CD166 and exhibited no difference between the two groups.
Conclusions:  It can be concluded that the use of Ca(OH)₂ or GIC as a lining material in indirect pulp capping procedures has the same effect on cells derived from the remaining dental pulp of exfoliated deciduous teeth which have responded favourably to the restorative treatments.     

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。     

人牙髓干细胞的体外培养和鉴定

目的　研究第三恒磨牙来源的人牙髓干细胞的表型和生物学性状。方法　从成人健康阻生牙中获取牙髓,酶消化法分离获得牙髓干细胞,计算细胞克隆形成率(CFU-F);免疫组化、RT-PCR法检测细胞的表面分子表达; 流式细胞仪测定细胞周期;体外分化诱导实验检测细胞的多向分化能力。结果　分离获得的牙髓干细胞在体外具有一定的克隆形成能力,诱导条件下部分牙髓干细胞可向脂肪、肌细胞和成牙本质细胞方向分化,符合干细胞的特征。结论　成功的从人第三恒磨牙牙髓中分离得到牙髓干细胞。     

Isolation and Characterization of Human Dental Pulp Stem/Stromal Cells From Nonextracted Crown-fractured Teeth Requiring Root Canal Therapy

IntroductionHuman dental pulp stem/stromal cells (hDPSCs) in adults are primarily derived from the pulp tissues of permanent third molar teeth in existing literatures, whereas no reports exist, to our knowledge, on deriving hDPSCs from a tooth without the need for surgical procedure. The aim of this study was to raise a novel idea to source hDPSCs from complicated crown-fractured teeth requiring root canal therapy.MethodshDPSCs were harvested from the pulp tissues for two complicated crown-fractured teeth requiring root canal therapy, retaining the teeth for subsequent prosthodontic rehabilitation, in a 41-year-old woman who had suffered a motorcycle accident. Pulp tissue from the left lower deciduous canine of a healthy 10-year-old boy (the positive control) was also removed because of high mobility and cultured for hDPSCs.ResultsThe hDPSCs derived from the two complicated crown-fractured teeth and the deciduous tooth were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages and also expressed stem cells markers and differentiation markers, which indicated their stem cell origin and differentiation capability. In addition, hDPSCs from both the complicated crown-fractured teeth and the deciduous tooth showed high expression for bone marrow stem cell markers including CD29, CD90, and CD105 and exhibited very low expression of markers specific for hematopoietic cells such as CD14, CD34, and CD45.ConclusionsThis report describes the successful isolation and characterization of hDPSCs from the pulp tissue of complicated crown-fractured teeth without tooth extraction. Therefore, pulp exposed in complicated crown-fractured teeth might represent a valuable source of personal hDPSCs.