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     

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。     

Transdentinal cytotoxicity of experimental adhesive systems of different hydrophilicity applied to ethanol-saturated dentin

The aim of this study was to evaluate the transdentinal cytotoxicity of experimental adhesive systems (EASs) with different hydrophilicity and dentin saturation solutions on odontoblast-like cells. One hundred 0.4-mm-thick dentin discs were mounted in in vitro pulp chambers and assigned to 10 groups. MDPC-23 cells were seeded onto the pulpal side of the discs, incubated for 48 h. The EASs with increasing hydrophilicity (R1, R2, R3 and R4) were applied to the occlusal side after etching and saturation of etched dentin with water or ethanol. R0 (no adhesive) served as controls. R1 is a non-solvated hydrophobic blend, R2 is similar to a simplified etch-and-rinse adhesive system and R3 and R4 are similar to self-etching adhesives. After 24 h, cell metabolism was evaluated by MTT assay (n = 8 discs) and cell morphology was examined by SEM (n = 2 discs). Type of cell death was identified by flow cytometry and the degree of monomer conversion (%DC) was determined by infrared spectroscopy (FTIR) after 10 s or 20 s of photoactivation. Data were analyzed by the Kruskal–Wallis and Mann–Whitney tests (α = 0.05). Dentin saturation with ethanol resulted in higher necrotic cell death ratios for R2, R3 and R4 compared with water saturation, although R2 and R3 induced higher SDH production. Photoactivation for 20 s significantly improved the %DC of all EASs compared with 10 s. A significant positive correlation was observed between the degree of hydrophilicity and %DC. In conclusion, except for R1, dentin saturation with ethanol increased the cytotoxicity of EASs, as expressed by the induction of necrotic cell death.     

TGF-β1对成牙本质细胞系MDPC-23中 Smads mRNA表达的影响

目的：研究TGF—β1对成牙本质细胞系MDPC-23细胞中Smads mRNA表达的影响，探讨成牙本质细胞内Smads分子基因表达调控机制。方法：培养MDPC-23细胞，细胞分为5组，分别用TGF—β1刺激0、0．5h、1．5h、4h、24h，提取总RNA。用半定量RT—PCR观察Smad2、Smad3和Smad7mRNA表达变化。结果：MDPC-23细胞表达Smad2、Smad3和Smad7 mRNA。在TGF—β1作用24h内，MDPC-23细胞内Smad2 mRNA表达水平无明显变化。在TGF—β1作用4h后，Smad3 mRNA表达水平下调。Smad7 mRNA水平在TGF—β1作用下1．5h达到最高峰，以后逐渐下降。结论：在成牙本质细胞内，TGF—β1可能通过不同的方式调控其细胞内信号分子Smad2、Smad3和Smad7的表达，从而使成牙本质细胞对Smad介导的TGF—β1信号得到精确调控。     

Smad8在成牙本质细胞系MDPC-23内BMP-2信号转导中的作用

目的：观察成牙本质细胞系MDPC-23内Smad8蛋白的表达,以及Smad8蛋白在骨形成蛋白-2（bonemorphogenetic protein-2,BMP-2）信号转导中的作用.方法：免疫组化法观察MDPC-23细胞内Smad8蛋白的表达,以及BMP-2和转化生长因子β1（TGF-β1）对MDPC-23细胞内Smad8分子定位的改变.结果：MDPC-23细胞的胞浆和胞核均有Smad8表达,在BMP-2刺激1 h后,Smad8从胞浆向胞核转位聚集.TGF-β1刺激后无类似现象发生.结论：成牙本质细胞系MDPC-23内存在Smad8的表达,且Smad8可特异性地将BMP-2的信号由胞浆转至胞核,但不能转导TGF-β1信号.     

Smad7在成牙本质细胞系MDPC-23内转化生长因子β信号转导中的作用

目的研究Smad7在成牙本质细胞系MDPC-23内转化生长因子(transforming growth factor-β1, TGF-β)信号转导的作用.方法培养MDPC-23细胞,免疫组化法观察TGF-β1对MDPC-23细胞内Smad7分子定位改变.通过瞬时共转染和报告基因检测方法观察Smad7分子对TGF-β1调控目的基因转录的影响.结果MDPC-23细胞表达Smad7蛋白,主要定位于细胞胞浆,在转化生长因子β1刺激30 min后,Smad7从胞核向胞浆转位聚集.TGF-β1显著诱导p3TP-Lux基础启动子活性.过表达Smad7完全抑制TGF-β1对p3TP-Lux启动子活性的诱导,而过表达Smad7反义cDNA载体则显著促进了TGF-β1对p3TP-Lux启动子活性的诱导.结论在成牙本质细胞系MDPC-23内,Smad7可能作为一种抑制性Smad分子在拮抗TGF-β1信号转导过程中发挥重要的作用.     

Early responses of human pulp to direct capping with resin adhesive systems and calcium hydroxide

Objective

Early responses of human pulp to Prime&Bond/phosphoric acid, Clearfil SE Bond, Clearfil S3 Bond and Dycal were investigated ex vivo.

牙本质基质蛋白1基因反义核酸对MDPC-23细胞碱性磷酸酶活性和矿化能力的影响

目的 :观察牙本质基质蛋白 1(dentalmatrixprotein ,DMP1)基因反义寡核苷酸 (AS -ODN)对成牙本质细胞系MDPC -2 3细胞碱性磷酸酶 (ALPase)活性和矿化能力的影响 ,深入研究成牙本质细胞的分化和牙本质形成的机制。方法 :以稳定表达DMP1的MDPC -2 3细胞为靶细胞、不同浓度DMP1反义核酸为阻断剂 ,观察不同作用时间对MDPC -2 3细胞ALPase活性的影响。用Westernblot法检测 10 μmol/LAS -ODN不同作用时间细胞DMP1表达情况 ,并观察连续作用 10d对该细胞矿化能力的影响。结果 :与正常组和S -ODN组比较 ,不同浓度的AS -ODN能够降低MDPC -2 3细胞ALPase活性 ,其中 10 μmol/LAS -ODN降低ALPase活性最为显著 (作用 5d时OD值最低 ,为 0 .3 3 78± 2 .0E)。Westernblot法检测到DMP1在MDPC -2 3细胞的表达在 10 μmol/LAS -ODN加入 12h后减弱 ,2 4h后完全阻断。此浓度AS -ODN连续作用细胞 10d后 ,vonKossa染色显示细胞中出现明显的钙盐沉积 ,矿化结节的数量和大小明显少于对照组。结论 :DMP1反义核酸能够降低MDPC -2 3细胞AL Pase活性和矿化能力 ,提示DMP1参与调节成牙本质细胞矿化过程 ,可能具有启动牙本质矿化的作用。     

Cytotoxic effects of dental cements on two cell culture systems

Abstract Four dental cements and one bis-GMA composite were tested in cultures of human periodontal ligament fibroblasts and mouse 3T3 fibroblasts. It was found that these cell types reacted to the cements with different intensities, and that there were differences in evaluation by phase contrast microscopy and succinic dehydrogenase histochemistry.     

Pulpal responses after application of current adhesive systems to deep cavities

The aim of the present study was to evaluate comparatively the pulpal tissue reactions of four adhesive systems placed in experimental cavities of healthy dog’s teeth. Class V cavities with a mean value of remaining dentin thickness (RDT) ranging between 0.55 ± 0.30–0.68 ± 0.38 mm were prepared. The cavities were treated with the following adhesive systems: Etch and Prime 3.0 (EP), Single Bond (SB), Clearfil SE Bond (CSE), and Prompt L-Pop (PLP). The pulpal tissue responses to dentin adhesives were assessed histopathologically at postoperative periods of 7, 21, and 65 days, and the results were subjected to statistical analysis. A significantly greater adverse inflammatory response was observed with the materials EP and PLP, while a significantly lesser degree of disorganization in the odontoblastic zone was found with the materials SB and CSE, in the postoperative period of 65 days. In addition, a thicker predentin zone was observed where SB material was applied. Application of the selected adhesive systems to non-exposed cavities, with an RDT, which ranged between the above-mentioned rates, was correlated with slight to moderate inflammation and odontoblast reduction depending on the materials used as well as upon the RDT.     

Cytotoxicity of current adhesive systems: In vitro testing on cell cultures of primary murine macrophages

Objectives

The aim of this study was to evaluate, in vitro, the potential cytotoxicity of dentinal adhesives on alveolar macrophages of Wistar rats, after diffusion through dentin.

Methods

The cytotoxicity of adhesives [single bond plus (SB), clearfil SE bond (CF) and Xeno V (XE)] applied to the occlusal surface of human dentin disks adapted to a dentin barrier test device were analyzed. The sets placed on a monolayer of cells were incubated for 24, 48 and 72 h. Culture medium and Escherichia coli lipopolysaccharides (LPS) were used as negative and positive controls, respectively. Cellular cytotoxicity was evaluated by observing the cell survival rate (MTT assay) and nitric oxide production (NO). The data were analyzed by one-way factorial ANOVA and Tukey's and Tamhane's paired comparisons T2 (α = 0.05).

Results

All the adhesive systems reduced the percentage of live cells by over 50%, compared with the control group. Within the same period of time, there was a statistically significant difference between the adhesives and LPS compared with the negative control group. SB presented a statistically significant difference between 24 h and 72 h, and XE between 48 h and 72 h. The quantity of NO produced in 24 h did not differ statistically between the NC and adhesive groups. After 48 h there was a significant difference between SB/CF and XE/NC. At 72 h only CF showed a significant difference from each of the other groups. LPS differed statistically from all the other groups at all the evaluation times.

Significance

Components of the adhesives tested may permeate the dentin in sufficient concentrations to cause death and damage to cell metabolism in the alveolar macrophages of rats, which indicates potential cytotoxicity to pulpal cells.     

牙本质基质蛋白1基因反义核酸对MDPC-23细胞内、外钙离子浓度的影响

目的观察牙本质基质蛋白1(DMP1)基因反义寡核苷酸作用下成牙本质细胞系MDPC-23细胞内、外钙离子浓度的动态变化,揭示牙本质矿化机制。方法以稳定表达DMP1的MDPC-23细胞为靶细胞,10μmol/L反义DMP1(AS-ODN)为阻断剂,用Western blot法检测不同时间细胞DMP1的表达情况,并观察不同作用时间内MDPC-23细胞内游离钙离子[(Ca2+)if]、总钙离子[(Ca2+)it]和细胞外钙离子[(Ca2+)e]的动态变化。结果Western blot法检测DMP1蛋白在MDPC-23细胞的表达在AS-ODN加入后12 h时减弱,24 h后完全阻断。与正常组和正义核酸组(S-ODN)相比较(平均荧光值为87.46±39.60),AS-ODN组(Ca2+)if先升高(平均荧光值12 h处为104.10±27.06;24 h处为98.46±19.92),AS-ODN作用24 h后,(Ca2+)if又降低(平均荧光值36 h处为77.54±14.95;48 h处为68.43±22.11);(Ca2+)it明显降低,于24 h处至最低值(0.142±0.233)mmol/L(P<0.01);(Ca2+)e呈上升趋势,且与对照组差异无显著性(P>0.05)。结论反义DMP1能够影响MDPC-23细胞内(Ca2+)if和(Ca2+)it浓度,提示DMP1参与调节成牙本质细胞的钙离子代谢和转运过程,可能在牙本质矿化过程中发挥作用。     

Response of human pulps capped with different self-etch adhesive systems

The aim of this study was to evaluate the response of human pulps capped with a calcium hydroxide hard-setting cement or with two-step self-etch adhesive systems. Pulp exposures were performed on the occlusal floor, and the bleeding control was performed with saline solution. The exposed pulp tissue was capped with Clearfil LB 2V (2V) or Clearfil SE Bond (SE) and restored with a composite resin. In control group, the pulpal wound was capped with Ca(OH)₂ cement and restored with Clearfil LB 2V or Clearfil SE Bond + composite resin. After 30 and 90 days, the teeth were extracted, processed for hematoxylin and eosin, and categorized in a histological score system. The pulpal response was worse for groups capped with the self-etch adhesive systems (2V and SE) in both periods of evaluation, when compared to their respective control groups at 90 days (p < 0.05). For both self-etch systems evaluated, the pulp tissue exhibited moderate to severe inflammatory cell infiltrate involving the coronal pulp with chronic abscesses. Dentin bridging was observed in a few specimens. For the calcium hydroxide groups, almost all specimens showed dentin bridge formation, with few scattered inflammatory cells and normal tissue below the pulp exposure site. Calcium hydroxide should be used as the material of choice for pulp capping, and the use of two-step self-etch adhesives for human pulp capping is contraindicated.     

神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白对牙髓细胞增殖的影响

目的探索神经生长因子受体介导的黑色素瘤抗原编码基因同源蛋白(NRAGE)对人牙髓细胞(h DPCs)和小鼠成牙本质细胞(MDPC-23)细胞增殖的影响。方法重组慢病毒转染细胞稳定敲除h DPCs和MDPC-23的NRAGE表达,体外组织块法原代培养h DPCs和MDPC-23,进而检测NRAGE对h DPCs和MDPC-23的增殖影响。采用CCK-8法分析NRAGE对h DPCs和MDPC-2细胞增殖的影响,流式细胞术分析NRAGE对h DPCs和MDPC-23的细胞周期分布和细胞凋亡影响。免疫荧光法检测NRAGE和NF-κB的表达和定位,分析NF-κB蛋白表达水平,并用IKK抑制剂处理细胞后,分析细胞周期和细胞凋亡。结果重组慢病毒转染后NRAGE的mRNA和蛋白水平下降显着。NRAGE敲减后抑制了h DPCs和MDPC-23的增殖活性和凋亡。NRAGE敲减后显示h DPCs的G0G1期滞留显著,而对MDPC-23没有影响。同时,NRAGE敲减后激活NF-κB信号通路。IKK抑制剂可以抑制NRAGE敲除后对h DPCs和MDPC-23的细胞凋亡的抑制作用。结论 NRAGE敲减后抑制牙髓细胞的增殖活性。NRAGE通过NF-κB信号通路调控h DPCs的细胞周期和凋亡。     

2种黏结系统与3种牙体硬组织间黏结界面的超微结构研究

目的:比较第7代自酸蚀黏结剂Adper Easy One与两步法全酸蚀黏结剂Adper Singlebond2黏结处理复合树脂充填后的形态学超微结构。方法:在20颗离体人磨牙的颊、舌侧制备盒形单面洞(直径5 mm,洞深3 mm),牙根中1/3处切取表面根片(长5mm,宽3mm),分为2组,每组10个牙冠、10个根片。对2组牙冠的窝洞和根片分别应用黏结剂Adper Singlebond2、Adper Easy One,充填黏结复合树脂后,将其从中间纵向剖开,各得到20个试件。每组随机抽取牙冠部和牙根部各10个试件,进行5000次冷热循环,剩余试件泡于蒸馏水中,常温存放1个月,砂纸打磨抛光,固定脱水,真空干燥、喷金,扫描电镜下观察剖面充填物边缘的黏结情况。结果:扫描电镜显示,全酸蚀Adper Singlebond2黏结处理、常温放置和冷热循环后,牙釉质、牙本质和牙骨质与黏结剂黏结紧密。自酸蚀黏结系统Adper Easy One黏结处理,常温下牙釉质黏结偶见细小裂隙,牙本质和牙骨质与黏结剂结合良好。冷热循环后,牙釉质黏结疏松,可见明显裂缝,牙本质和牙骨质黏结良好。结论:扫描电镜超微观结构观察显示,Adper Singlebond2全酸蚀黏结剂对牙釉质的黏结优于Adper Easy One自酸蚀黏结剂,而对于牙本质和牙骨质效果无明显差别。     

Effects of adhesive composite core systems on adaptation of adhesive post and cores under load

Objectives

To test marginal and internal adaptation of five different adhesive composite core systems under load.

Methods

30 human premolars were endodontically prepared and obturated with an epoxy sealer and vertically condensed gutta-percha. Thereafter the entire clinical crown was removed. The teeth were randomly assigned to five different composite core groups, all using the same fiber reinforced radicular post (DT White). Gr. 1: Optibond solo plus/Nexus II/Prodigy; Gr. 2: Scotchbond 1/Rely X Arc/Filtek P60; Gr. 3: EBS multi/Compolute applicap/Pertac II; Gr. 4: ART Bond/Parapost cement/Synergy; Gr. 5: Superbond C&B catalyst S + polymer/Metafil CX. Polyvinyl-siloxane impressions of the external margins of the cores were readied before and after 1,200,000 cycles of mechanical loading with max. 100 N at 45° to the longitudinal axis of the tooth. After loading, 5 of the 6 samples of each group were cut longitudinally and the sixth sample was cut transversally to be able to take replicas for evaluation of internal adaptation after loading.

Results

Percentages of external “continuous margins” ranged from 97.9 + −4.6 to 66.5 + −7.8 before and from 87.4 + −25.0 to 5.8 + −12.5 after loading. Internal adaptation ranged from 96.4 + −8.0 to 17.1 + −20.4 after loading for the core adaptation and from 89.8 + −12.2 to 65.9 + −14.3 for the dentin–luting composite interface and from 99.4 + −1.2 to 88.6 + −9.4 for the composite-post interface.

Conclusions

Surprisingly, the best materials’ combination for the adhesive composite core was a self-etch light cured adhesive with a chemically cured luting agent.     

Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

OBJECTIVE: This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. METHODS: A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). RESULTS: All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. SIGNIFICANCE: These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.     

Evaporating solvents with a warm air-stream: effects on adhesive layer properties and resin-dentin bond strengths

OBJECTIVES: This study evaluated the effect of a warm or cold air-dry stream for solvent evaporation on the microtensile resin-dentin bond strength (muTBS), nanoleakage pattern (SEM), degree of conversion (DC) and solvent evaporation rates (SE) of an ethanol/water- (Adper Single Bond, [SB] 3MESPE) and an acetone-based (Prime & Bond 2.1, [PB] Dentsply), two-step etch-and-rinse adhesive system. MATERIALS AND METHODS: Adhesives were applied on demineralized dentin surfaces. For SE, a warm or cold air-dry stream (10 s) was applied prior to light-activation (10 s). Bonded sticks (0.8mm2) were tested in tension (0.5 mm/min). Two bonded sticks from each tooth were immersed in a 50% (w/v) solution of silver nitrate (24 h), photodeveloped (8 h) and analyzed by SEM. The DC and solvent evaporation rate of the adhesives were evaluated under FTIR and analytical balance, respectively. Data were analyzed by two-way ANOVA and Tukey test (alpha=0.05). RESULTS: Higher muTBS and lower nanoleakage were observed when the SE step was performed with warm air-dry stream. However, the DC of the adhesives was not altered by the use of a warm air-dry. CONCLUSIONS: The use of a warm air-dry stream seems to be a clinical tool to improve the bond strength and the quality of the hybrid layer (less nanoleakage infiltration), since it might reduce the number of pores within the adhesive layer.