Smads蛋白在成牙本质细胞系MDPC-23中的表达及功能

目的研究Smads蛋白在成牙本质细胞系MDPC-23作为转化生长因子(TGF)β信号分子的作用。方法常规条件下培养MDPC-23细胞,在TGF-β1刺激培养1h后,观察细胞内Smad分子的定位变化。将Smads真核表达载体分别与报告基因载体p3TP-Lux瞬时共转染至MDPC-23,在TGF-β1刺激培养24h后,裂解细胞,用双荧光素酶报告基因检测系统检测细胞裂解液中的荧光素酶活性。结果MDPC-23细胞表达Smad2和Smad3蛋白分子,主要定位于细胞质,在TGF-β1刺激1h后,Smad2和Smad3从胞质向胞核转位聚集。TGF-β1可诱导p3TP-Lux基础启动子活性,约增加13倍。过表达野生型Smad3蛋白可促进TGF-β1对p3TP-Lux启动子活性的诱导,但是过表达Smad3突变体抑制TGF-β1对p3TP-Lux启动子活性的诱导。和Smad3作用相比,过表达Smad2野生型或突变型蛋白对TGF-β1诱导p3TP-Lux启动子活性无明显影响。结论在成牙本质细胞系MDPC-23内,Smad信号途径存在并参与介导TGF-β1诱导的转录调控。     

转录因子c-Jun和c-Fos在牙本质涎磷蛋白基因表达中的作用

目的 研究成牙本质细胞内转录因子c-Jun和c-Fos在牙本质涎磷蛋白（DSPP）基因转录调控中的作用, 探索成牙本质细胞内DSPP基因表达的调控机制。方法 细胞免疫组化观察MDPC-23细胞内c-Jun和c-Fos蛋白分子的表达。瞬时转染和报告基因检测c-Jun和c-Fos在DSPP基因转录中的作用。结果 MDPC-23细胞表达c-Jun和c-Fos蛋白,c-Jun和c-Fos主要分布在MDPC-23细胞胞核。c-Jun或c-Fos过表达均显著抑制DSPP基因启动子活性。结论 证实成牙本质细胞内转录因子c-Jun和c-Fos参与下调DSPP基因表达。     

小鼠牙本质涎磷蛋白启动子报告基因载体的构建和启动子活性分析

目的：克隆小鼠牙本质涎磷蛋自（dentin sialophosphopmtein,DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性。方法：细胞基因组提取，PCR，瞬时转染和报告基因检测。结果：从MDPC-23细胞基因组中克隆出长为1.5kbp的DSPP启动子，将启动子酶切成不同的片断，克隆到虫工业基础光素酶报告基因载体pC1.3-Enhancer,构建出4种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

小鼠牙本质涎磷蛋白5′端非编码区启动子报告基因载体的构建和分析

目的：克隆小鼠牙本质涎磷蛋白(dentin sialophosphopmtein，DSPP)基因启动子，构建含DSPP启动子不同片段的报告基因载体，在小鼠成牙本质细胞系MDPC-23中分析各种载体中DSPP启动子活性：方法：PCR、报告基因载体构建、瞬时转染和报告基因检测。结果：PCR获得DSPP启动子的3个不同片段，将它们克隆到萤火虫荧光素酶报告基因载体pG13-Enhancer，构建出3种含DSPP启动子不同片段的报告基因载体，将这些报告基因载体瞬时转染至MDPC-23细胞，载体中的启动子具有不同的活性。结论：成功构建了含小鼠DSPP启动子片段的报告基因载体，为以后研究DSPP基因表达调控的分子机制提供了实验工具。     

Smad分子在骨形成蛋白2调控成牙本质细胞Ⅰ型胶原基因表达中的作用

目的　研究Smad蛋白在骨形成蛋白 2 (bonemorphogeneticprotein 2 ,BMP 2 )调控小鼠成牙本质细胞系MDPC 2 3内Ⅰ型胶原α2链 [collagenα2 (Ⅰ ) ,COL1A2 ]表达中的作用。方法　细胞免疫组化观察MDPC 2 3细胞内BMP 2细胞内信号分子Smad1、Smad5和Smad6的表达。瞬时转染和报告基因检测观察Smad1、Smad5和Smad6在BMP 2调控COL1A2基因转录中的作用。结果　MDPC 2 3细胞表达Smad1、Smad5和Smad6。BMP 2能诱导含COL1A2基因启动子的荧光素酶报告基因活性。Smad1或Smad5过表达增强BMP 2诱导的COLIA2基因启动子活性 ,而Smad6过表达抑制BMP 2诱导的COL1A2基因启动子活性。过表达Smad1或Smad5突变型载体可以阻断BMP 2的诱导能力。结论　在MDPC 2 3细胞内 ,Smad信号途径存在并发挥功能 ,参与调控BMP 2对COL1A2基因的转录。Smad信号途径可能在BMP 2调控成牙本质细胞分化和牙本质形成过程中发挥重要作用     

核心结合因子α1对牙本质涎磷蛋白基因转录调控的影响

目的观察核心结合因子α1(corebindingfactorα1,cbfα1)对牙本质涎磷蛋白(dentinsialophosphoprotein,DSPP)基因启动活性的影响。方法选择小鼠成牙本质细胞样细胞MDPC-23为实验细胞,DSPP基因上游2.6kb片段(-2475bp～ 53bp)为启动子。通过采用瞬时转染、报告基因等方法,用SPSS10.0统计软件包对结果进行统计学分析,观察在MDPC-23中,cbfα1对DSPP基因启动子启动活性的影响。结果在MDPC-23细胞中,pGL3-Enhancer-2.6K pcDNA3-cbfα1共转染组荧光素酶的表达量显著小于pGL3-Enhancer-2.6K pcDNA3共转染组(P<0.01)。结论cbfα1对DSPP基因上游-2475bp～ 53bp区域的启动活性有抑制作用。     

RNA干涉法观察成纤维细胞生长因子18对成牙本质样细胞涎磷蛋白表达的影响

目的：通过RNA干涉的方法研究成纤维细胞生长因子18（Fibroblast Growth Factor18．FGF18）对成牙本质样细胞中牙本质涎磷蛋白（dentin sialophosphoprotein，DSPP）表达的影响。方法：RNA干涉质粒pH1-siFGF18瞬时转染成牙本质样细胞MDPC-23，RT-PCR检测FGF18和DSPP的表达，观察FGF18低表达对DSPP表达的影响。结果：瞬时转染RNA干涉质粒后，MDPC-23细胞内FGF18表达明显减少，与此同时DSPP的表达量也明显减少。结论：FGF18可以调控DSPP的表达，影响成牙本质细胞的分化。     

小鼠牙本质涎磷蛋白基因启动子的克隆与序列分析

目的 克隆小鼠牙本质涎磷蛋白(dentin sialophosphoprotein，DSPP)基因启动子片段。方法 培养MDPC一23细胞，从培养的细胞中提取基因组DNA，利用设计的上下游引物，进行PCR反应。将扩增得到的DSPP基因启动子片段克隆到pMD18-T栽体，酶切鉴定后，进一步进行DNA序列测定。结果 酶切结果表明成功构建重组质粒，序列分析结果与国外报道一致。结论 成功克隆获得小鼠DSPP基因的启动子片段。     

表皮生长因子对MDPC-23细胞增殖和ALP活性的影响

目的 :观察表皮生长因子 (epidermalgrowthfactor ,EGF)对成牙本质细胞系MDPC -2 3细胞增殖和碱性磷酸酶 (alkalinephosphatase ,ALP)活性的影响。 方法 :细胞培养、MTT比色测定法和碱性磷酸酶活性测定法。结果 :在一定浓度范围内 ,EGF明显促进MDPC -2 3细胞增殖 ,抑制MDPC -2 3细胞内碱性磷酸酶活性 ,并呈一定的剂量依赖性。结论 :EGF能促进成牙本质细胞增殖 ,而抑制其分化     

人牙胚发育过程中Smad2分子的表达与分布变化

目的 观察转化生长因子β（transforming growth factor－β，TGF－β）特异的细胞内信号转导分子Smad2在人牙胚发育过程中的表达与分布变化，探讨Smad2在牙发育过程中的作用。方法 制备人牙胚发育各期标本，免疫组化方法检测Smad2分子的表达。结果 Smad2在牙胚发育的各个时期存在特异的时空分布模式，其分布与TGF－β有相似之处。结论 首次观察到Smad2信号分子在人牙胚发育过程中的表达，Smad2作为TGF－β细胞内信号分子，可能参与上皮－间充质的信号诱导，同时也参与成釉细胞和成牙本质细胞的分化本质细胞原分化以及牙釉质和牙本质的形成。     

Dentin-specific proteins in MDPC-23 cell line

Only four established odontoblast-like cell lines have been reported in the literature (1–6). Of the four, only two synthesize dentin-specific proteins. These studies report that the cell line MO6-G3 synthesizes phosphophoryn (DPP), dentin sialoprotein (DSP) and dentin matrix protein-1 (DMP-1), while MDPC-23 synthesizes DSP, but not DMP-1. The objective of the present study was to determine whether polyclonal antibodies to rat DSP and DPP would label odontoblasts on microscopic sections of day-19 fetal mouse incisor odontoblasts as well as cultured cells of the MDPC-23 cell line. The spontaneously immortalized MDPC-23 cell line was derived from fetal mouse molar papillae, made continuous by the 3T6 method and cloned by dilution. These cultures have been passaged 77 times after cloning, form multilayered nodules, and have high alkaline phosphatase activity. The data show positive reactivity in odontoblasts in 19-d mouse fetal incisors as well as in cultures of MDPC-23 cells by fluorescence and confocal microscopy. In addition, these cultures were characterized by phase microscopy and scanning and transmission electron microscopy. These findings suggest that MDPC-23 cells are of the odontoblast lineage.     

肿瘤坏死因子受体相关分子6在成牙本质细胞中表达的研究

目的 :研究肿瘤坏死因子受体相关分子 6(TRAF6)是否在成牙本质细胞中表达。方法 :通过westernblot、免疫组化染色研究TRAF6蛋白在成牙本质样细胞系MDPC 2 3中的表达。结果 :Westernblot结果显示MD PC 2 3细胞总蛋白中有大小约 60Kda的TRAF6蛋白条带 ;免疫组化法表明TRAF6在MDPC 2 3细胞浆呈阳性表达。结论 :本实验从蛋白水平证实了成牙本质样细胞MDPC 2 3表达TRAF6,提示TRAF6可能是一种新发现的影响成牙本质细胞分化的胞内信号转导分子     

FGF18对成牙本质细胞样细胞增殖和碱性磷酸酶活性的影响

目的：研究FGF18对成牙本质细胞样细胞MDPC-23增殖和ALPase活性的影响。方法：用MTT法和酶动力法分别检测MDPC-23细胞经不同浓度的FGF18刺激48h和72h后细胞增殖和ALPase活性的变化。结果：在一定范围内,FGF18显著促进MDPC-23细胞的增殖和ALPase活性,随着浓度的增高和时间的延长,作用更加明显。结论：FGF18可能促进小鼠成牙本质细胞的增殖和分化。     

The roles of Smad 2/3 in transforming growth factor-beta 1 signaling in human dental pulp cells]

OBJECTIVE: To explore the roles of Smad 2/3 in transforming growth factor-beta(1) (TGF-beta(1)) signaling by human dental pulp cells. METHODS: Laser scanning confocal microscope was used to observe translocation of Smad 2/3 from plasma into nucleus in cultured dental pulp cells at early stage of TGF-beta(1) treatment, and changes of Smad 2/3 protein expression at later stage were evaluated by Western blot analyses. RESULTS: The expression of Smad 2/3 (fluorescence intensity) kept decreasing in cytoplasm but increasing in nucleus within 2 h after TGF-beta(1) treatment, forming a trend that Smad 2/3 translocated into nucleus from cytoplasma. The total amount of Smad 2 protein remained unchanged before and after TGF-beta(1) treatment, but the expression level of Smad 3 decreased markedly after 24 h treatment and kept dropping by 48 h. CONCLUSIONS: The results suggest that the Smad 2/3 may be the downstream signal transducers of TGF-beta(1) in human dental pulp cells and Smad 2/3 may mediate TGF-beta(1) signaling by translocation early in TGF-beta(1) treatment, while down-regulation of Smad 3 expression by TGF-beta(1) at later stage is involved in negative modulation of TGF-beta(1) signaling.     

Behavior of human dental pulp cells exposed to transforming growth factor-beta1 and acidic fibroblast growth factor in culture

The development of methods for regenerative endodontic procedures requires an understanding of the factors regulating the development of odontoblasts from adult cell populations such as pulpal cell lines. In this study, we exposed cultures of human pulp cells (7th passage) to growth factors including transforming growth factor-beta1 (TGF-beta1, at 1 or 5 ng/mL), acidic fibroblast growth factor (aFGF, 5 ng/mL), or a combination of the 2 growth factors and evaluated cellular morphology and markers of cell phenotype including alkaline phosphatase activity, osteocalcin, bone sialoprotein (BSP), and dentin sialophosprotein (DSPP). The mean number of nucleoli in the 1 ng/mL TGF-beta1 group was significantly higher than with 5 ng/mL aFGF. Alkaline phosphatase activity was significantly greater with 1 ng/mL TGF-beta1 versus 5 ng/mL TGF-beta1 + 5 ng/mL aFGF (P < .05). Osteocalcin mRNA was expressed in all samples. The cells exposed to 1 ng/mL TGF-beta1 were stimulated; however, exposure to growth factors for 8 days was not sufficient for expression of BSP and DSPP mRNA. Cells treated with 1 ng/mL TGF-beta1 exhibited higher activity, whereas 5 ng/mL aFGF-treated cells were inhibited. Although osteocalcin was observed in all cultures, suggestive of the potential for odontoblast formation, under the present conditions, the exposure to TGF-beta1 and aFGF was not sufficient to induce expression of the dentin matrix components BSP and DSPP.     

Transforming growth factor beta2 regulates growth and differentiation of pulp cells via ALK5/Smad2/3

Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.     

Cytotoxic effects of different concentrations of chlorhexidine

PURPOSE: To evaluate the cytotoxic effects of different concentrations of Chlorhexidine (Chx) to the odontoblast cell line MDPC-23. METHODS: The odontoblast-like cells were seeded (30,000 cells/cm2) in 60 wells of 24-well dishes and then incubated in contact with the following experimental and control solutions: Group 1: 0.0024% Chx; Group 2: 0.004% Chx; Group 3: 0.02% Chx; Group 4: Phosphate buffer saline solution (PBS, negative control); and Group 5: 0.06% H2O2 (positive control). Cell metabolic activity was measured by MTT assay and the cell morphology was analyzed by SEM. RESULTS: The cytotoxic effects of Chx are dose-dependent. The reduction in the cell metabolism for Groups 1, 2, and 3 was 24.8%, 29.9% and 70.8%, respectively. No statistical difference was observed between the Groups 1 and 2 in which no significant cell morphology changes occurred. Consequently, it was concluded that 0.02% Chx solution presents high cytotoxicity to the odontoblast-like cells MDPC-23. On the other hand, 0.0024% and 0.004% Chx causes slight cytopathic effects to the cultured cells.     

BMP2作用下人牙乳头细胞内Smad 1 mRNA表达的变化

目的：观察人牙乳头细胞内Smad1 mRNA的表达及在BMP2作用下，细胞内Smad1 mRNA的表达变化，探讨人牙乳头细胞内Smad1信号途径在BMP2调控牙乳头细胞分化中的作用。方法：原代培养的人牙乳头细胞用BMP2处理后，提取总RNA，采用Northern blot法，从mRNA水平观察Smad1基因的表达及含量变化。结果：从mRNA水平观察到Smad1基因在人牙乳头细胞内的表达，但在BMP2作用6、12、24h后，Smad1 mRNA表达量无显著变化。结论：人牙乳头细胞内存在Smad1信号转导途径，牙乳头细胞内Smad1 mRNA表达量不受BMP2调控。