牙周炎组织中miRNA表达谱差异筛选及功能预测分析

目的 筛选牙周炎患者组织中的差异表达miRNA,探讨其生物学功能以及参与的信号通路。方法 通过对微阵列数据库GSE54710中的158例牙周炎患者和40例健康人的牙龈组织中的基因芯片数据进行生物信息学分析,筛选差异表达miRNA,并预测参与的生物学功能和信号通路。采用SPSS 19.0软件包对数据进行统计学分析。结果 5种miRNAs（hsa-miR-451、hsa-miR-223、hsa-miR-486-5p、hsa-miR-3917、hsa-miR-671-5p）显著上调,4种miRNAs（hsa-miR-203、hsa-miR-210、hsa-miR-1246、hsa-miR-1260 ）显著下调。其中,hsa-miR-1260的靶基因584个,hsa-miR-451的靶基因139个。KEGG通路富集分析显示,hsa -miR-1260靶基因显著富集到TGF-beta等12条信号通路,hsa-miR-451靶基因显著富集到17条信号通路。结论 得到牙周炎组织中miRNAs的表达谱,牙周炎诱导的hsa-miR-1260和hsa-miR-451可能在牙周炎的生理病理学中起到关键作用。     

Notch配体Delta1对人牙髓干细胞分化的影响

目的:探讨Notch配体Delta1对体外人牙髓干细胞(dentalpulpstemcells,DPSCs)向成牙本质细胞分化能力的影响。方法:利用逆转录病毒载体建立高表达人Delta1基因的人牙髓干细胞系;实验分三组,正常牙髓干细胞组、转导细胞组及混合组(正常与转导细胞比例为100∶1),分别进行体外分化诱导。倒置显微镜观测各组细胞各生长期出现的时间;VonKossa染色计数各组形成钙化结节数;Westernblot法检测各组细胞牙本质涎磷蛋白表达。结果:与正常牙髓干细胞相比,转导细胞各生长期出现时间明显提前,形成的钙化细胞结节数目显著增加,牙本质涎磷蛋白表达显著升高。结论:Delta1基因转导牙髓干细胞仍保持了体外向成牙本质细胞分化的能力;Notch-Delta1信号与分化诱导因子协同作用可促进人牙髓干细胞向成牙本质细胞分化。     

CD146在人牙髓干细胞中表达的研究

目的:研究CD146在人牙髓干细胞及其诱导分化过程中的表达情况。方法:体外培养人牙髓干细胞,免疫荧光及流式细胞术检测CD146的表达。矿化诱导人牙髓干细胞分化,检测牙本质唾蛋白的表达,从mRNA及蛋白水平检测诱导过程中CD146的表达。结果:免疫荧光及流式细胞术证明人牙髓干细胞中CD146表达阳性。使用矿化诱导液培养人牙髓干细胞,通过检测到牙本质唾蛋白的表达,证明细胞已向成牙本质细胞方向分化;在此诱导过程中,CD146在人牙髓干细胞中的表达逐渐下调。CD146在人牙髓干细胞中有较特异性的表达,有可能作为其特异性标志物。     

小分子整联蛋白结合配体N-连接糖蛋白家族成员在牙髓干细胞分化过程中的作用

体外诱导牙髓干细胞向成牙本质细胞分化并观察其过程,是目前研究牙髓干细胞分化机制和寻找其分化标志的主要方法。在成牙本质细胞分化和矿化过程中,小分子整联蛋白结合配体N-糖蛋白(SBLING)家族成员发挥着重要作用。下面就SBLING家族成员牙本质涎磷蛋白、牙本质基质蛋白-1和细胞外基质磷酸糖蛋白在牙髓干细胞分化过程中的作用作一综述。     

人牙髓细胞向成牙本质细胞分化的蛋白质组学研究

目的 采用蛋白质组学方法分析人牙髓细胞向成牙本质细胞分化过程中细胞蛋白表达谱的改变,揭示特征蛋白在分化过程中所起的作用.方法对人牙髓细胞进行矿化诱导,提取诱导前后细胞总蛋白,双向电泳分离蛋白质,DeCyder V6.0软件确定差异蛋白点,质谱鉴定差异蛋白质.结果双向电泳确认46个差异蛋白质斑点,质谱鉴定20个蛋白斑点,差异蛋白质涉及细胞周期调节、能量代谢、信号传导等细胞生物学过程.结论蛋白质组学技术可高通量筛选与人牙髓细胞向成牙本质细胞分化相关的功能蛋白.     

血管生成素4对牙髓干细胞成牙本质向分化的作用

目的 探讨血管生成素4(angiopoietin 4,ANGPT4)对牙髓干细胞成牙本质向分化的影响。方法 本实验研究已通过单位伦理委员会审查批准,并获得患者知情同意。将人前磨牙进行固定、脱钙、脱水、包埋和切片,免疫荧光染色观察ANGPT4的表达及定位。体外分离培养人牙髓干细胞（human dental pulp stem cells,hDPSCs）,于倒置相差显微镜下观察hDPSCs的生长状态及形态;流式细胞术检测细胞表面相关分子标志物的表达;碱性磷酸酶和茜素红S染色鉴定h DPSCs成牙本质向分化的潜能;油红O染色鉴定hDPSCs成脂向分化潜能。提取hDPSCs成牙本质向诱导后不同时间点的RNA,采用RT-qPCR分析hDPSCs体外成牙本质向分化过程中ANGPT4基因及成牙本质细胞相关基因的表达。采用siRNA基因沉默技术沉默hDPSCs中ANGPT4基因的表达,通过RT-qPCR和Western blot检测沉默效率;在hDPSCs中沉默ANGPT4基因表达24 h后进行成牙本质向诱导,在诱导的第7天和14天分别进行碱性磷酸酶和茜素红S染色,检测沉默ANGPT4后hDPSCs的...     

经典Wnt通路调节骨髓间充质干细胞向成牙本质样细胞的分化

目的:探讨经典Wnt通路在骨髓间充质干细胞向成牙本质样细胞分化中的作用,以期为临床治疗牙髓损伤提供新策略。方法:用牙胚细胞条件培养液诱导骨髓间充质干细胞向成牙本质样细胞的分化,培养液内分别加入外源性Wnt激活剂Wnt3a/抑制剂DKK-1,检测经典Wnt通路中β-catenin、LEF1、TCF4及成牙本质样细胞特异表达物DSPP、DMP-1变化。结果:骨髓间充质干细胞经Wnt激活剂/抑制剂处理后,胞浆内β-catenin水平明显上升/下降,其下游核内转录因子LEF1、TCF4基因水平明显上升/下降,而细胞内DSPP、DMP-1的表达明显降低/升高。结论:Wnt信号通路抑制骨髓间充质干细胞向成牙本质样细胞分化。     

人牙髓干细胞及非牙髓干细胞中微小RNA表达谱比较研究

目的 比较微小RNA（miRNAs）在人牙髓干细胞（dental pulp stem cells, DPSCs）及非DPSCs中的表达差异,探讨其在维持DPSCs干性状态中的作用。方法 本研究于2013年1—10月在福建医科大学附属口腔医院完成。 原代培养人牙髓细胞,利用结合了STRO-1特异性抗体的免疫磁珠分选获得DPSCs,并进行成牙本质样细胞的诱导分化,检测碱性磷酸酶（ALP）、骨钙素（OC）值以及进行von Kossa染色,鉴定其分化能力。采用miRNA基因芯片技术,检测DPSCs和非DPSCs中miRNAs的表达,筛选出差异表达的miRNAs。结果 与非DPSCs相比,DPSCs中表达上调超过2倍的miRNAs有11个,表达下调超过2倍的miRNAs有3个。结论 miRNAs表达谱的变化可能与DPSCs干性状态的维持相关。     

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目的 比较微小RNA（miRNAs）在人牙髓干细胞（dental pulp stem cells, DPSCs）及非DPSCs中的表达差异,探讨其在维持DPSCs干性状态中的作用。方法 本研究于2013年1—10月在福建医科大学附属口腔医院完成。 原代培养人牙髓细胞,利用结合了STRO-1特异性抗体的免疫磁珠分选获得DPSCs,并进行成牙本质样细胞的诱导分化,检测碱性磷酸酶（ALP）、骨钙素（OC）值以及进行von Kossa染色,鉴定其分化能力。采用miRNA基因芯片技术,检测DPSCs和非DPSCs中miRNAs的表达,筛选出差异表达的miRNAs。结果 与非DPSCs相比,DPSCs中表达上调超过2倍的miRNAs有11个,表达下调超过2倍的miRNAs有3个。结论 miRNAs表达谱的变化可能与DPSCs干性状态的维持相关。     

成牙本质细胞特异分子标志物的研究进展

牙髓干细胞是一类存在于牙髓组织中,保持着高度的增殖和分化潜能,受到刺激后能向终末细胞分化的细胞。在牙髓干细胞的研究过程中,常需要根据成牙本质细胞分子标记物的表达来判断牙髓干细胞的分化进程。随着研究的进展,可供选择的成牙本质细胞分子标记物也越来越广泛。下面就成牙本质细胞分化的特异分子标记物的研究进展作一综述。     

miR-103对牙髓干细胞增殖及其向成牙本质细胞分化的影响

目的:探讨miR-103对牙髓干细胞向成牙本质细胞分化的影响.方法:从即刻拔除的成入第三磨牙中分离培养人牙髓干细胞,将培养的细胞分为对照组、miRNA阴性对照组、miR-103过表达组和miR-103降低表达组,利用CCK-8法检测人牙髓细胞的增殖能力变化,利用q-PCR和Western Bolt技术检测人牙髓细胞中R...     

TWIST1 promotes the odontoblast-like differentiation of dental stem cells

Stem cells derived from the dental pulp of extracted human third molars (DPSCs) have the potential to differentiate into odontoblasts, osteoblasts, adipocytes, and neural cells when provided with the appropriate conditions. To advance the use of DPSCs for dentin regeneration, it is important to replicate the permissive signals that drive terminal events in odontoblast differentiation during tooth development. Such a strategy is likely to restore a dentin matrix that more resembles the tubular nature of primary dentin. Due to the limitations of culture conditions, the use of ex vivo gene therapy to drive the terminal differentiation of mineralizing cells holds considerable promise. In these studies, we asked whether the forced expression of TWIST1 in DPSCs could alter the potential of these cells to differentiate into odontoblast-like cells. Since the partnership between Runx2 and Twist1 proteins is known to control the onset of osteoblast terminal differentiation, we hypothesized that these genes act to control lineage determination of DPSCs. For the first time, our results showed that Twist1 overexpression in DPSCs enhanced the expression of DSPP, a gene that marks odontoblast terminal differentiation. Furthermore, co-transfection assays showed that Twist1 stimulates Dspp promoter activity by antagonizing Runx2 function in 293FT cells. Analysis of our in vitro data, taken together, suggests that lineage specification of DPSCs can be modulated through ex vivo gene modifications.     

Zinc chloride for odontogenesis of dental pulp stem cells via metallothionein up-regulation

Introduction

Previous studies have shown that zinc chloride (ZnCl₂) can induce metallthionein (MT) in the liver and kidney to protect tissues against toxicants and shows a better corneal wound healing than conventional drugs do. We hypothesized that ZnCl₂ can promote odontogenesis of dental pulp stem cells (DPSCs) via MT. The purpose of this study was to investigate the effects of ZnCl₂ on human DPSCs and the expression of MT.

Methods

DPSCs were isolated by flow cytometry with selective surface marker CD146 and STRO-1. After they grew into confluence, DPSCs were induced into odontoblasts with or without ZnCl₂ supplemented in the culture medium for 21 days. The effect of ZnCl₂ on DPSCs differentiation was examined followed by alkaline phosphatase staining/activity and quantitative real-time polymerase chain reaction analysis.

Results

By treating DPSCs with ZnCl₂, the duration of mineralization was shortened and expressions of differentiation markers into odontoblasts were more significant than those without ZnCl₂ stimulation. Besides, the MT gene expression was increased with the increasing expressions of odontoblasts’ markers after treated with ZnCl₂.

Conclusion

This was the first report that ZnCl₂ could promote odontoblastic differentiation of DPSCs through the up-regulation of gene MT.     

牙髓干细胞分化过程中L型钙离子通道羧基末端的表达

目的:探讨牙髓干细胞(DPSCs)分化过程中L型钙离子通道羧基末端的表达。方法:利用酶消化法体外分离、培养大鼠牙髓干细胞;吉姆萨染色法检测大鼠牙髓干细胞的克隆形成能力;神经诱导体系下诱导牙髓干细胞向神经样细胞分化,免疫荧光染色检测细胞分化后胶质纤维酸蛋白(glial fibrillary acidic pro-tein,GFAP)的表达和细胞分化前后L型钙离子通道Cav 1.2及羧基末端的表达。结果:牙髓干细胞的克隆形成能力为每1 000个细胞形成2～17个克隆;免疫荧光染色检测诱导后细胞GFAP表达阳性;免疫荧光染色检测显示:牙髓干细胞分化前L型钙离子通道Cav 1.2羧基末端表达于细胞膜上,细胞分化后羧基末端同时表达于细胞膜上和细胞核中。结论:L型钙离子通道Cav 1.2羧基末端在牙髓干细胞分化过程中发生核转位,羧基末端可能在牙髓干细胞的分化过程中发挥着一定的作用。     

Inhibition of Delta1 promotes differentiation of odontoblasts and inhibits proliferation of human dental pulp stem cell in vitro

Objective

Dental pulp stem cells (DPSCs) have been receiving more attentions recently as an important biomaterial for tissue engineering. Notch signalling plays a key role in regulating self-renewal and differentiation of a variety of cells. The objective of this study is to investigate the effects of Notch-Delta1 RNA interference (RNAi) on the proliferation and differentiation of human dental pulp stem cells in vitro.

Design

In the present study, we performed gene knockdown of Notch ligand Delta1 in DPSCs using lentivirus-mediated Delta1-RNAi. Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, Cell viability assay (CCK-8) and Western blot analysis of proliferating cell nuclear antigen (PCNA). Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of cells was detected with Alkaline phosphatase ALP activity assay, Alizarin red S staining, calcium concentration measurement, and Western blot analysis of Dentine sialophosphoprotein (DSPP).

Results

Lentivirus-mediated Delta1-RNAi stably knocked-down the expression of Delta1 and Notch signalling, and some of DPSCs/Delta1-RNAi displayed changes in morphology or DSPP expression. The growth rate of Delta1-deficient DPSCs was significantly suppressed as compared with wild type DPSCs and control lentivirus vector transfected DPSCs. Furthermore, the differentiating capability of DPSCs/Delta1-RNAi into odontoblasts is much higher than the two control groups.

Conclusions

Notch signalling plays a crucial role in regulating self-renewal and differentiation in DPSCs. The deficient Notch signalling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation-inducing conditions. These findings suggested that DPSCs/Delta1-RNAi might be applicable to stem cell therapies and tooth tissue engineering.     

miR‐140‐5p‐mediated regulation of the proliferation and differentiation of human dental pulp stem cells occurs through the lipopolysaccharide/toll‐like receptor 4 signaling pathway

Human dental pulp stem cells (DPSCs) are oral mesenchymal stem cells with potential to differentiate into various cell types. Recent studies of DPSCs have focused on microRNAs (miRNAs), a class of small noncoding RNAs that play crucial roles in regulating DPSC phenotypes. In the current study, the expression of miR‐140‐5p was significantly decreased during lipopolysaccharide (LPS)‐mediated differentiation of DPSCs in vitro. Overexpression of miR‐140‐5p enhanced proliferation of DPSCs and inhibited DPSC differentiation, whereas suppression of miR‐140‐5p produced the opposite effect. Moreover, the expression of toll‐like receptor 4 (TLR‐4), a critical regulator of DPSCs, was negatively correlated with the levels of miR‐140‐5p. A luciferase reporter analysis confirmed that miR‐140‐5p could regulate TLR‐4 by directly binding to the 3′‐untranslated region (3′‐UTR) of the TLR4 mRNA. Additionally, we suppressed TLR‐4 expression by treating cells with a TLR‐4 inhibitor, CLI‐095, and demonstrated that the effect of the miR‐140‐5p inhibitor on DPSC proliferation and differentiation could be partially reversed by blocking TLR‐4. Taken together, our data suggest that miR‐140‐5p is a novel miRNA that regulates DPSC proliferation and differentiation.     

Inhibition of human dental pulp stem cell differentiation by Notch signaling

Notch signaling plays a critical role in development and cell fate specification. Notch receptors and ligands have been found to be expressed in dental epithelium or mesenchyme in the developing tooth, suggesting that Notch signaling may regulate odontogenesis. Post-natal human dental pulp stem cells (DPSCs) isolated from the dental pulp have characteristics of mesenchymal stem cells and can differentiate into odontoblasts. In this study, we examined whether Notch signaling regulated the odontoblastic differentiation of DPSCs. We found that over-expression of the Notch ligand, Jagged-1, activated the Notch signaling pathway in DPSCs. Jagged-1 inhibited the odontoblastic differentiation of DPSCs in vitro. Jagged-1-expressing DPSCs could not form mineralized tissues in vivo. Moreover, over-expression of the constitutively activated Notch1 intracellular domain (Notch-ICD) also inhibited odontoblastic differentiation of DPSCs. Taken together, our results demonstrate that Notch signaling can inhibit the odontoblastic differentiation of DPSCs.