低浓度β-甘油磷酸钠培养牙髓干细胞向成牙本质细胞分化及相关因子的表达

背景:以往研究用来诱导成牙本质细胞分化的培养基普遍借鉴成骨细胞的诱导浓度,其他浓度的诱导情况尚无相关研究。目的:观察在低浓度β-甘油磷酸钠条件培养下,牙髓干细胞向成牙本质细胞分化过程中,牙本质基质蛋白1、牙本质涎蛋白和细胞外基质磷酸糖蛋白的表达情况。方法:分离培养人牙髓干细胞,用不同浓度的诱导液诱导牙髓干细胞分别向脂肪细胞,成骨细胞分化,证实其多向分化能力。用5 mmol/Lβ-甘油磷酸钠诱导培养牙髓干细胞向牙本质细胞分化,分别在培养第7,14,21,28天提取各组细胞RNA,反转录PCR检测牙本质基质蛋白1、牙本质涎蛋白和细胞外基质磷酸糖蛋白的表达情况;诱导牙髓干细胞向成牙本质细胞分化形成的矿化结节用Alizarin Red S法检测。结果与结论:人牙髓干细胞经过诱导可证实其成功分化为脂肪细胞和成骨细胞;反转录PCR结果显示,5 mmol/Lβ-甘油磷酸钠培养牙髓干细胞在培养7,14,21 d,各组细胞牙本质基质蛋白1、牙本质涎蛋白的mR NA表达增加,伴随细胞外基质磷酸糖蛋白表达下调;培养至28 d,行矿化结节检测发现牙髓干细胞向成牙本质细胞成功矿化,可见红染的矿化结节。结果证实,5 mmol/Lβ-甘油磷酸钠诱导培养的牙髓干细胞可成功分化为成牙本质细胞,并下调细胞外基质磷酸糖蛋白mR NA的表达,同时上调牙本质基质蛋白1及牙本质涎蛋白mR NA的表达。     

脂多糖改变牙髓干细胞的生物学特性

背景:龋病发生时,包括细菌在内的各种刺激沿牙本质小管传导至牙髓,牙髓组织中的牙髓干细胞受到刺激后增殖迁移至受损的牙本质下方,形成修复性牙本质以补充和修复受损组织。在此过程中,脂多糖作为细菌的主要毒力因子,是否可以影响牙髓干细胞增殖、迁移及成牙本质向分化等生物学行为,还有待研究。目的:观察脂多糖对牙髓干细胞增殖能力、迁移能力和成牙本质向分化能力的影响。方法:组织块酶消化法培养牙髓干细胞,给予0,0.1,1,10 mg/L脂多糖刺激后,采用MTT法检测牙髓干细胞增殖情况,划痕实验和Transwell实验检测牙髓干细胞的迁移能力;给予1 mg/L脂多糖和矿化诱导液刺激21 d后,茜素红染色检测细胞矿化结节形成能力,RT-PCR检测成牙本质相关基因的表达。结果与结论:①0.1,1,10 mg/L脂多糖组的吸光度值在第1,3,5,7天4个时间点均低于0 mg/L脂多糖组(P<0.01),迁移能力均高于0 mg/L脂多糖组;②牙髓干细胞矿化诱导21 d后,1 mg/L脂多糖刺激组所形成的矿化结节数量和面积明显小于0 mg/L脂多糖组(P<0.01),成牙本质相关基因OCN、BSP、ALP表达量显著低于0 mg/L脂多糖组(P<0.01);③结果表明,脂多糖刺激牙髓干细胞后其增殖能力和成牙本质向分化能力降低,迁移能力明显增强。     

人牙髓细胞向成牙本质细胞诱导分化中细胞周期变化

目的建立成牙本质细胞样细胞体外培养体系，观察体外培养的牙髓细胞向成牙本质细胞分化过程中细胞周期的变化。方法利用组织块酶解法培养人牙髓细胞并进行鉴定，使用矿化诱导液诱导其向成牙本质细胞样细胞分化。对细胞增殖情况使用流式细胞仪进行细胞周期测定。结果①获得的牙髓细胞均为波形丝蛋白阳性，角蛋白阴性，证明为中胚层来源的细胞。②诱导分化的牙髓细胞在2周左右进入复层生长期；3周左右开始有细胞结节形成．周围细胞呈放射状排列，部分细胞出现细长突起并呈极性排列：4周左右细胞团中央Von Kossa染色为阳性。③在诱导开始后1周，细胞处于活跃的增殖期，以后细胞增殖变慢．3周后多数细胞处于G0G1期。结论实验成功建立了成牙本质细胞样细胞体外培养体系，所获得的成牙本质细胞样细胞具有成牙本质细胞的部分形态和生物学功能，诱导分化后细胞增殖变慢．是研究成牙本质细胞的较好模型。     

Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制

目的 探讨Chir99021对大鼠牙髓干细胞(DPSCs)成骨诱导分化的影响及机制。方法 原代分离大鼠DPSCs,利用荧光免疫法进行验证。设置不同浓度的Chir99021,通过CCK-8检测细胞增殖情况挑选最适浓度。利用茜素红染色验证细胞成骨诱导分化。最后通过Real-time PCR、Western blotting检测成骨诱导分化相关基因与蛋白无翅型MMTV整合位点家族成员1(Wnt1)、无翅型MMTV整合位点家族成员3A(Wnt3a)、β-连环蛋白(β-catenin)、轴抑制蛋白2抗体(Axin2)、骨钙素(OCN)、牙本质基质蛋白1(DMP1)的mRNA和蛋白表达情况。结果 牙本质涎磷蛋白(DSPP)和干细胞标记物波形蛋白(vimentin)阳性表达,说明大鼠DPSCs分离成功。大鼠DPSCs成骨诱导后钙沉积物显著上升,加入糖原合成酶激酶-3β(GSK-3β)的小分子抑制剂Chir99021后钙沉积物明显减少。大鼠DPSCs成骨诱导分化后Wnt1、Wnt3a、β-catenin、Axin2、OCN和DMP1表达明显上升,而加入Chir99021后Wnt1、Wnt3a、β-ca...     

脂多糖调控Notch 信号通路作用于人牙髓干细胞增殖、分化、凋亡的实验研究

目的:探讨脂多糖调控Notch 信号通路对人牙髓干细胞增殖、分化、凋亡的影响及机制。方法:从牙髓组织中分离出人牙髓干细胞(hDPSCs),CCK8 实验检测0、0.1、1、10 μg/ ml 的脂多糖处理hDPSCs 1、3、5、7 d 后细胞的增殖情况;RT-PCR 检测1 μg/ ml 的脂多糖处理hDPSCs 0、3、7、14、21 d 后矿化相关基因ALP、DSPP、DMP1 的mRNA 表达情况;流式细胞仪检测1 μg/ ml 的脂多糖处理hDPSCs 0、7、14、21 d 后的细胞凋亡情况;Western blot 检测Cleaved caspase3、Notch1、Hes1 的蛋白表达。结果:不同浓度的脂多糖刺激hDPSCs 1、3、5 d 后细胞的增殖均无显著差异,培养至第7 天时,0.1、1、10 μg/ ml 的脂多糖组细胞的增殖均显著低于0 滋g/ ml 的脂多糖组(P<0.01);脂多糖处理hDPSCs 3 d 时ALP、DSPP、DMP1 的mRNA 表达与对照组比较差异均无统计学意义(P>0.05),7、14、21 d 时ALP、DSPP、DMP1 的mRNA 表达均显著高于对照组(P<0.01);脂多糖处理hDPSCs 7、14、21 d 时细胞的凋亡率及Cleaved caspase3、Notch1、Hes1 蛋白表达均显著高于对照组(P<0.01),21 d 时ALP、DSPP、DMP1 的mRNA 表达及细胞凋亡率和Cleaved caspase3、Notch1、Hes1 蛋白表达均有下降趋势。结论:脂多糖可降低hDPSCs 的增殖,促进其矿化和凋亡,其机制与激活Notch 信号通路有关。     

人牙髓细胞骨基质蛋白表达的研究进展

骨基质蛋白在骨骼和牙齿形成，以及损伤修复过程中起着重要作用。本对骨涎蛋白(BSP)、骨桥蛋白(OPN)、牙本质涎蛋白(DSP)、牙本质磷蛋白(DPP)、牙本质涎磷蛋白(DSPP)的表达，以及在牙齿形成、损伤修复，对组织工程中成骨组织作用等研究进展进行了概述。     

尼古丁抑制人牙髓干细胞增殖和分化

目的探讨尼古丁的刺激对人牙髓干细胞增殖、分化的影响。方法培养人牙髓干细胞,流式细胞计量术鉴定细胞表面抗原;用不同浓度的尼古丁(10-4、10-3和10-2 mol/L)刺激牙髓干细胞,培养0、1、2、3和4 d后,CCK8法检测细胞增殖能力;茜素红染色法检测细胞分化过程中矿化结节形成,RT-qPCR和免疫印迹(Western blot)检测牙本质涎磷蛋白(DSPP)、碱性磷酸酶(ALP)、骨桥素(OPN)及细胞外信号调节激酶(ERK)、c-Jun氨基末端激酶(JNK)、p38(p-ERK、p-JNK、p-p38)等MAPK通路相关蛋白表达。结果培养第3、4天时,与对照组相比,尼古丁刺激时A值显著降低(P<0.05);与对照组相比,尼古丁刺激时矿化结节形成数、DSPP、ALP、OPN mRNA和蛋白、p-ERK、p-JNK、p-p38蛋白表达显著降低(P<0.05)。结论尼古丁抑制人牙髓干细胞增殖、成骨分化能力。     

人骨髓间充质干细胞体外成骨与成脂分化过程中FGF1及其受体表达的变化

目的 研究人骨髓间充质干细胞(hBMSCs)成骨与成脂分化过程中成纤维细胞生长因子1(FGF1)及其受体(FGFRs)的动态表达,探讨FGF1在hBMSCs成骨与成脂分化过程中的作用.方法 密度梯度离心法分离hBMSCs,取第3代细胞分别进行成骨和成脂诱导分化并染色鉴定.通过real-time PCR方法检测成骨和成脂标志基因以及FGF1/FGFRs的表达,免疫荧光染色法检测诱导分化前后FGF1的表达.Real-time PCR及油红O染色鉴定外源性FGF1对hBMSCs成骨与成脂分化的影响.结果 体外诱导hBMSCs成骨和成脂分化后茜素红和油红O染色结果呈阳性;FGF1和FGFRs在诱导过程中呈动态表达;免疫荧光染色结果表明FGF1主要在细胞质中分布,成骨诱导3d后在细胞核中的表达增加;相比对照组,加入外源的FGF1组7d后脂滴数量明显减少,成脂标志基因表达受到抑制(P＜0.01),而加入外源的FGF1 3 d骨桥蛋白(OPN)表达升高(P＜0.05).结论 FGF1能够抑制hBMSCs的成脂分化,并且可能通过促进骨桥蛋白表达而提前hBMSCs的成骨矿化.     

转化生长因子β3诱导人乳牙牙髓干细胞向成牙本质样细胞的分化

背景：转化生长因子β3与肝素联合作用后对人乳牙牙髓干细胞的增殖和矿化能力的作用目前仍有待研究。目的：探讨转化生长因子β3对人乳牙牙髓干细胞向成牙本质样细胞分化的作用。方法：采用酶消化法将人乳牙牙髓分离培养,获得人乳牙牙髓干细胞;对体外培养的第3代人乳牙牙髓干细胞单独加入25 μg/L重组转化生长因子β3、10 U/mL肝素,或者二者联合进行培养;通过Q-PCR和Western-blotting方法,分别检测乳牙牙髓干细胞中牙本质涎磷蛋白基因及牙本质涎蛋白表达的情况;通过碱性磷酸酶试剂盒检测乳牙牙髓干细胞碱性磷酸酶活性的改变。结果与结论：乳牙牙髓干细胞在诱导体系中生长状态良好。25 μg/L转化生长因子β3+10 U/mL肝素联合作用组的碱性磷酸酶活性明显增强,与转化生长因子β3单独作用组、肝素单独作用组以及对照组相比差异有显著性意义(P < 0.01);转化生长因子β3+肝素联合作用组的茜素红染色呈强阳性,Q-PCR和Western-blotting结果均显示,转化生长因子β3+肝素联合作用组的牙本质涎磷蛋白mRNA和蛋白表达均明显升高。结果可见在转化生长因子β3与肝素联合作用的诱导下,可促进乳牙牙髓干细胞分化为成牙本质样细胞。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

改良酶消化法分离培养人乳牙牙髓干细胞

背景：乳牙牙髓干细胞具有极强的增殖活力,可以在短期内获得组织工程需要的细胞量。 目的：比较传统酶消化法和改良酶消化法获取人乳牙牙髓干细胞的成功率及生物学特性。 方法：取无龋滞留乳切牙12 颗,随机数字表法均分为两组,分别应用传统酶消化法和改良酶消化法分离获取乳牙牙髓细胞,在不同代次细胞中检测细胞扩增天数、收获量、生长曲线、倍增时间、克隆形成率、细胞表面特异标记物STRO-1、CD146、CD34 和CD45 的表达。细胞成骨、成脂诱导分化能力及通过检测碱性磷酸酶和牙本质涎蛋白表达水平,判定细胞牙向分化潜能。 结果与结论：两组细胞生长曲线,扩增天数、细胞收获量及细胞倍增时间相比,差异有显著性意义(P <0.05);牙向分化实验结果显示,改良酶消化法碱性磷酸酶和牙本质涎蛋白较传统酶消化法和对照组而言呈强阳性表达。然而,两组在克隆形成率、细胞表面特异标记物及多项诱导分化等方面差异无显著性意义(P > 0.05)。说明改良酶消化法与传统酶消法相比,可在较短时间内完成同源乳牙牙髓干细胞体外扩增培养,可为牙齿再生治疗提供高质量种子细胞,是乳牙牙髓干细胞体外培养的可靠方法。     

Odontogenic differentiation of human dental pulp stem cells induced by preameloblast-derived factors

The differentiation of odontoblasts is initiated by the organization of differentiating ameloblasts during tooth formation. However, the exact roles of ameloblast-derived factors in odontoblast differentiation have not yet been characterized. We investigated the effects of preameloblast-conditioned medium (PA-CM) on the odontogenic differentiation of human dental pulp stem cells (hDPSCs) in?vitro and in?vivo. Furthermore, we analyzed the PA-CM by liquid chromatography-mass spectrometry to identify novel factors that facilitate odontoblast differentiation. In the co-culture of MDPC-23 cells or hDPSCs with mouse apical bud cells (ABCs), ABCs promoted differentiation of odontoblastic MDPC-23 cells and facilitated odontoblast differentiation of hDPSCs. PA-CM, CM from ABCs after 3 days culture, was most effective in increasing the dentin sialophosphoprotein promoter activity of odontoblastic MDPC-23 cells. When PA-CM-treated hDPSCs were transplanted into immunocompromised mice, they generated pulp-like structures lined with human odontoblast-like cells showing typical odontoblast processes. However, during recombinant human bone morphogenenetic protein 2-treated hDPSCs transplantation, some of the cells were entrapped in mineralized matrix possessing osteocyte characteristics. After proteomic analyses, we identified 113 types of proteins in PA-CM, of which we characterized 23. The results show that preameloblast-derived factors induce the odontogenic differentiation of hDPSCs and promote dentin formation.     

Human dental pulp stem cells differentiate into neural crest-derived melanocytes and have label-retaining and sphere-forming abilities

Adult tissues contain highly proliferative, clonogenic cells that meet criteria of multipotent stem cells and are potential sources for autologous reparative and reconstructive medicine. We demonstrated that human dental pulp contains self renewing human dental pulp stem cells (hDPSCs) capable of differentiating into mesenchymal-derived odontoblasts, osteoblasts, adipocytes, and chondrocytes and striated muscle, and interestingly, also into non-mesenchymal melanocytes. Furthermore, we showed that hDPSC cultures include cells with the label-retaining and sphere-forming abilities, traits attributed to multipotent stem cells, and provide evidence that these may be multipotent neural crest stem cells.     

细胞力学机械刺激对牙髓干细胞的影响

文题释义：牙髓干细胞：通过GRONTHOS等对牙髓细胞的研究,发现一种具有与间充质干细胞相似免疫表型和形成矿化结节能力的细胞,称为牙髓干细胞,具有自我更新、多向分化和较强的克隆能力。细胞力学：研究细胞在力学载荷作用下细胞膜和细胞骨架的变形、弹性常数、黏弹性、黏附力等力学性质,以及机械因素对细胞生长、发育、成熟、增殖、分化、衰老和死亡等的影响及其机制研究,是生物力学的一个前沿领域,也是组织工程学的一个重要组成部分。  摘要背景：力学刺激对于机体内很多器官、组织的发育和损伤修复发挥重要的调控作用。除生物化学因素外,细胞力学等机械因素越来越被认为是影响牙髓干细胞行为和功能的关键调节因素。目的：综述细胞力学刺激对牙髓干细胞生物学行为的作用及影响。方法：通过检索PubMed、Embase、Medline、CNKI数据库,分别以“牙髓干细胞,机械压力,机械张力,剪切力,压应力,细胞增殖,成骨分化”为中文检索词和“human dental pulp stem cells(hDPSCs),mechanical strain,mechanical stretch,mechanical tension,shear stress,cell proliferation,osteogenesis differentiation”为英文检索词进行检索,选取与细胞力学机械刺激参与影响牙髓干细胞增殖、分化的56篇文献进行综述。结果与结论：细胞力学机械刺激是影响细胞增殖、分化、蛋白表达和凋亡的重要生物学因素。牙髓干细胞是牙髓组织中的间充质干细胞,其生物学行为也受到细胞力学机械刺激的影响。细胞力学刺激参与牙髓干细胞的增殖、成牙/成骨分化,并且当牙本质受到流体流动力作用时,会激活其机械感受器来调节并维持牙齿的完整性。介导牙髓干细胞生物学行为的信号通路包括MAPK、Wnt、Akt及BMP-7、Nrf2/HO-1等,通过调控这些信号通路进而对牙髓干细胞增殖及成牙/成骨分化发挥不同程度的促进及抑制作用。ORCID: 0000-0001-6191-6539(李峻青) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

Natural mineralized scaffolds promote the dentinogenic potential of dental pulp stem cells via the mitogen-activated protein kinase signaling pathway

The selection of a suitable scaffold material is important for dentin tissue regeneration, as the characteristics of biomaterials can potentially influence cell proliferation and differentiation. We compared the effects of different scaffolds on dentin regeneration based on dental pulp stem cells (DPSCs) and investigated the regulatory mechanisms of odontogenic differentiation of DPSCs by these scaffolds. Five different scaffolds were tested: demineralized dentin matrix (DDM), ceramic bovine bone (CBB), small intestinal submucosa (SIS), poly-L-lactate-co-glycolate, and collagen-chondroitin sulfate-hyaluronic acid. DPSCs cultured on DDM and CBB exhibited higher levels of alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein, osteocalcin, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1) than those cultured on the other three scaffolds. Further, the phosphorylation levels of mitogen-activated protein kinase (MAPK) ERK1/2 and p38 in DPSCs cultured on DDM and CBB were also significantly enhanced compared with the other three scaffolds, and their inhibitors significantly inhibited odontogenic differentiation as assessed by ALP activity and mRNA expression of DSPP and DMP-1. The implantation experiment confirmed these results and showed a large amount of regular-shaped dentin-pulp complex tissues, including dentin, predentin, and odontoblasts only in the DDM and CBB groups. The results indicated that natural mineralized scaffolds (DDM and CBB) have potential as attractive scaffolds for dentin tissue-engineering-promoted odontogenic differentiation of DPSCs through the MAPK signaling pathway.     

Effect of ITGA5 down-regulation on the migration capacity of human dental pulp stem cells

Background: The purpose of this study was to evaluate the role of integrin-α5 (ITGA5) in regulating the migration capacity of human dental pulp stem cells (hDPSCs), which might provide new evidence for understanding the repair and regeneration mechanisms of dental pulp tissues. Materials and methods: The enzyme digestion method was employed to isolate the hDPSCs from dental pulp tissues. The cell surface markers of hDPSCs were detected using flow cytometry analysis. Then the colony forming and multi-differentiation capacity of hDPSCs were evaluated. The lentivirus vector that carried the ITGA5 shRNA was constructed and real-time PCR was used to examine the effectiveness of ITGA5 shRNA lentivirus. Then transwell assay was performed to evaluate the impact of ITGA5 inhibition on the migration capability of hDPSCs. Results: Our results showed that the cells we isolated from the dental pulps were positive for mesenchymal stem cells biomarkers. In addition, the cells possessed both colony forming capacity and multi-differentiation potential. ITGA5 shRNA lentivirus could not only infect hDPSCs with high efficiency, but also down-regulate the expression level of ITGA5 mRNA significantly (P<0.01). The transwell assay revealed the number of cells that migrated to the lower chamber was significantly less in the ITGA5 shRNA group compared with that in the scrambled shRNA group (P=0.016). Conclusion: ITGA5 plays an important role in maintaining and regulating the normal migration capacity of hDPSCs.     

Odontogenic differentiation and dentin formation of dental pulp cells under nanobioactive glass induction

Bioactive glass (BG) has been widely used in bone regeneration; however, reports on the biological effects of BG on dental pulp cells are rare. This study aims to investigate the effects of nanoscale BG (n-BG) on odontogenic differentiation and dentin formation of dental pulp cells and to compare these effects with those of microscale BG (m-BG). Human dental pulp cells (hDPCs) from third molars were cultured directly with m-BG and n-BG in vitro. The cell proliferation increased at 0.1 mg ml⁻¹ BG, which also had a chemotactic effect on hDPCs. The mineralization capacity and expression of odontogenic-related proteins and genes (dentin sialophosphoprotein, dentin matrix protein 1 and collagen type I) of hDPCs were significantly up-regulated under BG induction, and were particularly higher in the n-BG group than in the control group. m-BG and n-BG combined with pulp tissues were transplanted into the dorsum of immunodeficient mice to observe their biological effects on dental pulp cells in vivo. A continuous layer of dentin-like tissue with uniform thickness, a well-organized dentinal tubule structure and polarizing odontoblast-like cells aligned along it was generated upon the n-BG layer, whereas some irregular sporadic osteodentin-like mineralized tissues were observed in the control group. This study reveals that BG, especially n-BG, induces the odontogenic differentiation and dentin formation of dental pulp cells and may serve as a potential material for pulp repair and dentin regeneration.