TLR9在成牙本质细胞中的表达

目的：研究成牙本质细胞中TLR9、DSPP基因表达特征及信号转导途径。方法：采用RT-PCR检测TLR9在小鼠牙髓组织中及TLR9、DSPP在成牙本质细胞系中的表达。用CpGODN-A和CpGODN-B刺激细胞,在时间梯度0、3、6、9、12、24h,检测TLR9、DSPP基因的表达特征。结果：TLR9mRNA在小鼠牙髓组织中有表达。TLR9、DSPPmRNA在成牙本质细胞中都有显著的表达,在CpGODN刺激下显著上调,在6h处于峰值。结论：TLR9在成牙本质细胞中有表达;TLR9的特异性表达对DSPP基因表达量有影响。     

基质金属蛋白酶-2，9，8在成人成牙本质细胞中的表达及转移生长因子对其分泌的影响

目的:观察研究基质金属蛋白酶(matrix metalloproteinases,MMPs)在成牙本质细胞中的表达和调控.方法:利用酶谱、Western blot和免疫组化等方法,研究MMP-2,9,8的表达以及转移生长因子β1(Transforming growth factor beta 1,TGF-β1)作用后MMP-2,9,8的表达变化.结果:酶谱显示MMP-2、MMP-9在成牙本质细胞中均有表达,TGF-β1上调MMP-2下调MMP-9;Western blot显示成牙本质细胞中有MMP-8的表达,TGF-β1增强其表达;免疫组化检测到MMP-8定位表达在成牙本质细胞层.结论:成牙本质细胞是MMP-2,9,8的来源之一;TGF-β1可能调节MMP-2,9,8的表达.     

永生化成牙本质细胞表达牙本质基质蛋白的实验研究

目的　探讨永生化人成牙本质细胞样细胞系hTERT-hOd-l表达牙本质基质蛋白的情况。方法　矿化液培养hTERT-hOd-l细胞5周,检测骨钙素(OC)分泌量和碱性磷酸酶(ALP)活性。采用免疫组织化学、RT-PCR和原位杂交方法检测Ⅰ型胶原、骨涎蛋白(BSP)、牙本质基质蛋白1 (DMP1)以及成牙本质细胞标志物牙本质涎磷蛋白(DSPP) 和牙本质涎蛋白(DSP)在细胞中的表达。结果　在矿化液诱导下,hTERT-hOd-l细胞ALP活性和OC分泌量升高。 hTERT-hOd-l细胞在mRNA水平上表达BSP、DMP1和DSPP,在蛋白质水平上表达DSP和Ⅰ型胶原。结论　hTERT- hOd-l细胞在体外表达牙本质基质蛋白,具有矿化的潜能。     

成牙本质细胞的基本特征及其研究进展

成牙本质细胞作为牙髓细胞的主要成分，具有在牙发育期间和成熟牙内生成牙本质的功能。目前的研究多基于牙髓细胞的二维体外培养，随着对牙髓组织研究的不断深入，牙髓细胞体外三维立体模型的建立将是未来研究的热点。作者回顾了近年来对成牙本质细胞的研究情况，主要阐述了成牙本质细胞的形态、超微结构、功能、蛋白表达的特征及其研究进展和今后的研究方向。     

Toll样受体4在人成牙本质细胞和牙髓组织中的表达

目的 探讨人牙髓组织和成牙本质细胞中Toll样受体4（TLR4）的表达与分布特点，及其在牙髓组织天然免疫防御中的可能作用。方法从新鲜拔除的人第三磨牙获取牙髓组织和成牙本质细胞，扫描电镜观察牙髓摘除后髓腔壁成牙本质细胞形态和附着情况；RT—PCR检测人牙髓组织以及成牙本质细胞中TLR4mRNA的表达情况：采用免疫印迹技术和免疫组织化学方法检测TLR4蛋白的表达与定位。结果扫描电镜观察牙髓摘除后成牙本质细胞大部存留在髓室壁上，细胞形态良好：RT-PCR结果发现健康牙髓、成牙本质细胞均可表达TLR4mRNA，产物大小为278bp；免疫印迹法检测正常牙髓、成牙本质细胞在相对分子质量89000附近出现蛋白条带；免疫组化显示牙髓组织中的成牙本质细胞、血管内皮细胞TLR4免疫反应阳性。正常牙髓中牙髓成纤维细胞不表达TLR4。结论成牙本质细胞通过表达TLR4参与牙髓牙本质复合体的先天免疫反应，TLR4在牙髓抵御外来病原微生物入侵的宿主免疫防御中起重要作用。     

细胞外基质磷酸化糖蛋白在人牙髓细胞成牙本质分化中的表达

义(P<0.05).蛋白质印迹法检测显示各矿化标记的蛋白表达均较对照组上调.结论 在hDPC诱导成牙本质分化的过程中,MEPE与DSPP、DSP、BSP、Ⅰ型胶原呈现相似的表达变化趋势,提示MEPE与hDPC的成牙本质分化有关,可作为hDPC向成牙本质样细胞分化的标志.     

人成牙本质细胞的分离和鉴定

目的:分离并鉴定完整的成熟人成牙本质细胞。方法:收集人健康恒牙,采用胶原酶和蛋白酶联合消化法分离出成牙本质细胞,并对分离出的细胞进行细胞形态学和免疫组织化学鉴定。结果:实验分离出的细胞为柱状或立方状,有较长的细胞突起,具有典型的成牙本质细胞形态,免疫组化染色显示细胞表达Ⅰ型胶原、DMP1和DSP蛋白。结论:本研究分离得到了完整的成熟人成牙本质细胞,为后期研究成牙本质细胞的分化、功能及特点奠定基础。     

诱导成牙本质样细胞在体内外的矿化研究

目的：人早期胚胎颌突外胚间充质细胞在体外能被诱导分化为成牙本质样细胞，表达牙本质特异的涎磷蛋白。本研究探讨该诱导分化细胞在体内外矿化的可能。方法：转化生长因子-βl(TGF-β1)和牙本质非胶原蛋白(DNCP)作用于三维培养的外胚间充质细胞，诱导10d，然后将诱导的成牙本质样细胞接种于裸鼠皮下，X线照相，组织切片，HE染色，观察细胞在体内的矿化情况。同时消化细胞，培养于矿化液中，观察细胞在体外的矿化能力。结果：8周后，裸鼠皮下接种的胶原细胞密度明显高于对照组。5周，胶原内的细胞为单极长突起；8周时，细胞周围可见牙本质样基质结构沉积。矿化液中的细胞3d出现复层生长，2ld，VonKossa染色阳性。结论：诱导的成牙本质样细胞在体内可以形成类似牙本质样基质，在体外能形成矿化结节，为有功能的终末分化的成牙本质细胞。     

Par3在成牙本质细胞胞间连接及其生物学功能中的作用研究

目的:通过下调分离缺陷基因3(partitioning defective-3,Par3)的表达探究其对小鼠成牙本质细胞系细胞(odontoblast lineage cells, OLCs)胞间连接的调控作用及相关生物学影响。方法:通过免疫荧光实验分别观察Par3在大鼠牙髓组织及小鼠成牙本质细胞中的表达及分布;利用Western blot检测炎症状态下成牙本质细胞Par3的表达情况;利用小干扰RNA(siRNA)敲低成牙本质细胞Par3的表达后,通过透射电镜观察Par3表达下调前后小鼠成牙本质细胞胞间连接结构的变化;利用Western blot及细胞免疫荧光技术检测Par3表达下调前后细胞紧密连接相关分子ZO-1(zonula occludens-1)的表达及分布情况;最后,通过EdU(5-ethynyl-2’-deoxyuridine)、Transwell及流式细胞术等检测Par3表达下调对成牙本质细胞增殖、迁移及凋亡等生物学功能的影响。结果:Par3在大鼠牙髓组织中与成牙本质细胞分布一致。体外条件下Par3表达于成牙本质细胞胞质内,且在炎症状态下表达水平下降。Par3表达下调后小...     

不同龋深状态下人成牙本质细胞的凋亡及Bcl-2,Bax的表达

目的探讨龋病进展中人成牙本质细胞的凋亡情况及可能机制。方法选择即刻拔除的第三磨牙80颗,正常牙、浅龋、中龋和深龋各20颗。HE染色、TUNEL染色和免疫组化染色后观察龋损部位下方成牙本质细胞的凋亡情况及Bcl-2、Bax的表达。结果①中龋以后,成牙本质细胞数量明显减少,凋亡指数随龋病的进展而逐步升高。②正常牙组和浅龋组Bcl-2及Bax的表达水平均较低,中龋组和深龋组两蛋白表达水平均显著升高,但Bax的升高幅度更显著,Bcl-2/Bax蛋白比值降低。结论随着龋损的进展,成牙本质细胞凋亡增多,Bcl-2/Bax蛋白比值变化是调控其凋亡的重要机制。     

基质金属蛋白酶MMP-2在龋病发展过程中的表达研究

目的：观察基质金属蛋白酶MMP-2在健康及不同程度龋损牙齿牙髓中的表达，初步研究MMP-2与龋病发展的关系。方法：以健康、浅龋和深龋牙齿中牙髓组织为研究对象，利用免疫组化、RT—PCR、实时荧光定量PCR和Western印迹检测MMP-2在其中的表达差异。采用单因素方差进行统计学分析。结果：组织标本免疫组化显示MMP-2在健康组和浅龋组中呈弱阳性表达，在深龋组中则表达强阳性。在健康组中仅表达于成牙本质细胞中，而在龋病组中牙髓细胞亦有表达。实时荧光定量PCR和Western印迹均检测到MMP-2在深龋组中表达明显高于其他两组，健康组和浅龋组表达无明显差异。结论：MMP-2的表达部位和强度与龋病的进展相关。     

MMP1 and MMP20 contribute to tooth agenesis in humans

Objective

Variations in genes that are critical for tooth formation may contribute to the tooth agenesis. MMPs are potential candidate genes for dental alterations based on the roles they play during embryogenesis. The aim of this study was to investigate the possible association between MMP1, MMP3, and MMP20 and tooth agenesis.

Methods

One hundred sixty-seven nuclear families from two different populations were analysed, 116 from Brazil and 51 from Turkey. Probands had at least one congenitally missing tooth. DNA samples were obtained from blood or saliva samples and genotyping was performed using TaqMan chemistry. In addition, Mmp20 was selected for quantitative real-time polymerase chain reaction analysis with SYBR Green I Dye in mouse tooth development.

Results

Associations between tooth agenesis and MMP1 (p = 0.007), and MMP20 (p = 0.03) were found in Brazilian families. In the total dataset, MMP20 continued to be associated with tooth agenesis (p = 0.01). Mmp20 was not expressed during the initial stages of tooth development.

Conclusion

Our findings provide evidence that MMP1 and MMP20 play a role in human tooth agenesis.     

Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA microarray and two-dimensional gel electrophoresis

Complementary DNA (cDNA) microarray and two-dimensional (2-D) gel electrophoresis, combined with mass spectrometry, enable simultaneous analysis of expression patterns of thousands of genes, but their use in pulp biology has been limited. Here we compared gene and protein expression of pulp tissues from sound and carious human teeth using cDNA microarray and 2-D gel electrophoresis to evaluate their usefulness in pulp biology research and to identify the genes with changes in carious teeth. The cDNA microarray revealed several differentially expressed genes and genes with a high expression in both tissues. These genes have various functions, e.g. effects on vascular and nerve structures, inflammation, and cell differentiation. Variability between cDNA hybridizations indicates that the overall gene expression pattern may vary significantly between individual teeth. The 2-D gel electrophoresis revealed no change between healthy and diseased tissue. The identification of 96 proteins in the pulp tissue revealed none of the gene products with corresponding high/different mRNA expression in cDNA microarray. Interestingly, we detected also a hypothetical protein (putative nucleoside diphosphate kinase), and present therefore the first evidence for the existence of this protein. Even though the methods reveal potentially important gene expression, they may currently have only limited value in in vivo pulp biology research.     

Retention of sealants over carious and sound tooth surfaces

Bacteriologic and clinical studies suggest that sealants can be used therapeutically on incipient caries of the enamel. The present study was designed to explore this approach further by determining whether there is a difference in retention rates on sound or carious tooth surfaces sealed for the first time as well as resealed surfaces. A total of 1766 teeth were sealed for subjects 12-14 yr of age of which 120 were judged to be carious. Mouths were divided sagittally on a random basis and an ultraviolet light activated resin with a filler or an autopolymerizing resin without a filler was placed on contralateral surfaces of all molar and premolar teeth. The effects of sealant type, caries status, arch and tooth type on retention over a 2-yr period were tested using Mantel-Haenszel statistics. At the end of 1 yr, 88% of the teeth sealed with the autopolymerizing resin were judged to be completely intact compared to 82% of the teeth filled with the ultraviolet light resin. At the end of the second year the retention rates were 84% and 75% respectively. The retention rates for carious and sound teeth at the end of both years were almost identical. Overall, premolars had a 15% more favorable retention rate than molars. There were minimal differences between arches although maxillary premolars had approximately a 5% higher retention rate than mandibular premolars. The reverse was true for molar teeth. Retention of sealants at the end of the second year (resealed after 1 yr) was 4% less than comparable teeth sealed initially.     

Chemical analysis of sound and carious enamel of permanent tooth

Enamel consists mainly of inorganic material (96%) and only a small amount of organic substance and water (4%). The inorganic material is similar to apatite. The originally found apatite mineral remains basically unchanged except at the surface in contact with the oral tissues, where diffusion processes operate. Enamel reflects the trace element environment present in the tissue fluids at the time of tissue development. These are variations of types and concentration of inorganic elements found in permanent and deciduous enamel. Variations are also seen in sound and carious enamel.