大鼠成釉细胞的原代培养和光、电镜观察

目的 通过对大鼠成釉细胞进行分离培养,为在离体状态下研究成釉细胞的发育和影响因素以及与釉质发育的关系奠定实验基础。方法 运用组织细胞分离培养技术分离大鼠的成釉细胞并进行原代培养,在光、电镜下观察其形态结构。结果 成釉细胞成簇生长,胞体多呈柱状,胞浆内细胞器发育完好。结论 原代培养的成釉细胞基本保持其在体时的形态及结构特征,此种方法可以用于成釉细胞的体外研究。     

氟对体外培养大鼠成釉细胞活性和细胞凋亡的影响

目的 观察氟对体外培养的大鼠成釉细胞活性的影响,为探讨氟斑牙的形成机制提供依据。方法 取对数生长期的大鼠成釉细胞,在培养液中加入浓度为0、0.4、0.8、1.6、3.2、6.4 mmol/L的 NaF溶液,培养24、48、72 h后,采用CCK-8检测各组细胞的活性。荧光显微镜下观察成釉细胞核形态的变化,流式细胞术分析氟对细胞凋亡的影响。采用SPSS13.0软件包对数据进行统计学分析。结果 ①NaF浓度为0.4、0.8 mmol/L时,对成釉细胞有促增殖作用;NaF浓度为1.6、3.2、6.4 mmol/L时,对成釉细胞的活性有抑制作用,随着NaF浓度的增加,对细胞的抑制作用也逐渐增强,并且这种双向调节作用呈时间依赖性。②NaF浓度为0.4、0.8 mmol/L时,鲜见核破碎;NaF浓度为1.6、3.2 mmol/L时,存在核破碎,并且随着浓度的提高,核破碎的数量随之增加,即1.6 mmol/L浓度的NaF可引起成釉细胞凋亡,随着浓度的增加,细胞凋亡的数量随之增加。结论 ①氟对体外培养成釉细胞的增殖具有双向调节作用,即低浓度促进,高浓度抑制。②浓度超过1.6 mmol/L时,NaF诱导成釉细胞凋亡。     

人胚成釉细胞体外培养的实验研究

目的 :探索成釉细胞离体培养方法 ,建立成釉细胞体外模型。方法 :运用组织细胞培养技术分离人胚成釉细胞进行原代培养 ,倒置显微镜下观察培养细胞生长特点 ,免疫组织化学染色检测培养细胞釉原蛋白。结果 :培养细胞成片生长 ,细胞形态结构清楚。免疫组化染色 ,多数细胞抗釉原蛋白抗体染色阳性。结论 :用消化培养法 ,胶原做基质饲养层 ,含 5 %小牛血清的HAMF -12作培养基并加入EGF、INS、HC ,可用于人成釉细胞的体外培养     

氟对体外培养大鼠成釉细胞 HAT-7细胞活性和对细胞内 Ca2+浓度的影响

目的:观察氟对体外培养的大鼠成釉细胞HAT-7细胞活性及细胞内Ca2+浓度的影响。方法:取对数生长期的HAT-7细胞,分别加入不同浓度的Na F培养液,培养24、48、72 h后,用CCK-8检测细胞的活性;流式细胞术分析氟对细胞凋亡的影响;激光共聚焦显微镜检测细胞内钙离子的浓度。结果:Na F浓度为0.4、0.8 mmol/L时,促进成釉细胞增殖;当Na F浓度大于1.6 mmol/L时,促进成釉细胞凋亡,Na F浓度为1.6 mmol/L时,细胞早期凋亡数量增加,激光共聚焦显微镜检测证实,1.6mmol/L Na F可以诱导大鼠成釉细胞内Ca2+浓度升高,Na F对HAT-7细胞的上述作用与浓度和作用时间呈正相关。结论:低浓度氟促进HAT-7细胞增殖,高浓度则抑制其增殖。Na F浓度超过1.6 mmol/L时,可诱导成釉细胞凋亡,并诱导成釉细胞内Ca2+浓度增加。     

氟化物诱导成釉细胞发生内质网应激的机制

氟斑牙是在牙齿发育阶段机体摄入过量的氟所引起的牙釉质发育不全,为慢性氟中毒病初期最典型的症状,其流行趋势具有一定的地域性,常发生于6岁前有高氟区定居史的患者.内质网是机体蛋白质加工、合成、运输的重要场所,也是细胞内重要的钙储存库之一.当细胞处于病毒感染、营养剥夺、钙超载、氧化应激等环境时,会造成内质网功能障碍,折叠蛋白...     

不同浓度氟对大鼠切牙成釉细胞Smad2/3表达的影响

目的:研究不同浓度氟对大鼠切牙生长过程中成釉细胞内信号转导分子Smad2/3表达的影响,探讨氟斑牙的发病机制。方法:40只Wistar大鼠随机分为3组,建立氟斑牙动物模型。用HE染色和免疫组化方法观察氟对大鼠切牙成釉细胞形态及Smad2/3表达的影响,采用MetaMorph显微图像分析系统和SPSS12.0软件包分别进行图像和数据分析。结果:给氟组大鼠切牙出现典型氟斑牙症状。给氟组Smad2/3的表达显著低于对照组(P<0.01),但低氟组和高氟组间未见显著差异(P>0.05)。结论:过量氟可能通过抑制Smad2/3的表达来影响釉质的分化和发育,导致氟牙症的发生。     

钟状期成釉细胞的体外生长研究

目的:通过对乳牙牙胚成釉细胞的体外培养生长情况的研究,了解成釉细胞的生长和分化过程.方法:解剖分离因疾病原因引产遗弃的19周胎儿下颌骨,半侧固定后拍摄数字X线影像,连续切片HE染色;另半侧未固定的组织分离乳牙牙胚成釉器,胶原酶消化法制成细胞悬液,采用差速贴壁和无血清培养基选择性培养成釉细胞.培养的乳牙胚成釉细胞通过细胞角蛋白14免疫细胞染色鉴定,并以RT-PCR方法检测釉质基质蛋白的表达.结果:X线影像显示牙胚冠部有硬组织形成,组织学乳牙牙胚处于钟状期,可见分化的成釉细胞分泌大量细胞外基质,釉牙本质界处釉质已矿化.无血清培养基中牙源性上皮的生长分化良好,细胞角蛋白14染色阳性,RT-PCR显示有釉蛋白、釉原蛋白和成釉蛋白的表达.结论:采用无血清培养基可以选择性的培养乳牙胚钟状期成釉细胞,能够较好反映成釉细胞的生长分化状态.钟状期分化的成釉细胞进入细胞外基质分泌活跃期,并在釉牙本质界处最早出现釉质的矿化.     

不同时期成釉细胞氟损伤程度差异的动物实验研究

目的：GRP78和BCL-2在氟中毒大鼠不同时期成釉细胞中的表达和分布,探索氟牙症发病机制。方法：选择20只Wistra大鼠,饲喂氟浓度为75mg F-/L的自来水,8周后处死,通过HE染色和免疫组化技术观察大鼠成釉细胞形态和GRP78和BCL-2在氟中毒大鼠不同时期成釉细胞中的表达。结果：GRP78在成釉细胞分泌前期和分泌期表达高于转换期和成熟期,BCL-2在分泌期和转换期表达最高,在分泌前期和成熟期表达下降,各组间具有显著统计学差异（P<0.05）.结论：分泌早期和分泌期成釉细胞对氟诱导的内质网更加敏感,而在分泌期和转换期其抗凋亡能力更强。     

大鼠成釉细胞离体培养研究

目的：探讨成釉细胞离体培养方法及其特性。方法：采用贴壁培养法培养大鼠成釉细胞，用倒置显微镜观察细胞形态，用免疫组化检测细胞合成釉原蛋白的情况，用电镜观察成釉细胞离体培养时的形态。结果：大鼠成釉上皮细胞经消化酶解后原代培养于明胶包被的培养皿上，细胞贴壁良好，仅有少量成纤维细胞。在培养细胞生长一定时间后，主要为上皮型细胞成片生长，在细胞传至Ｆ２代时仍能分泌釉原蛋白。传至Ｆ５代时，细胞逐渐转变为长梭形，类似成纤维细胞。扫描电镜可见细胞突起，并有基质分泌。结论：用明胶作基质，对大鼠成釉细胞作离体培养，可以较长时间地维持其细胞特性。     

T-2毒素对大鼠切牙成釉细胞凋亡的影响

目的:研究不同剂量T-2毒素对大鼠切牙成釉细胞凋亡的影响.方法:选择30只大鼠,随机分为3组,对照组、实验组1(100ng/gBW·d-1),实验组2(200ng/gBW·d-1).4周后处死大鼠,利用原位细胞凋亡检测技术(TUNEL)观察T-2毒素作用下大鼠切牙成釉细胞的凋亡现象.结果:T-2毒素染毒组成釉细胞凋亡率...     

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目的探讨氟斑牙的发病机制及褪黑素是否对氟斑牙的发生有拮抗作用。方法 2009年3—10月于中国医科大学公共卫生学院将40只Wistar大鼠随机分为6组,包括阴性对照组、低氟组、高氟组、低氟+褪黑素组、高氟+褪黑素组。建立氟斑牙动物模型,制作切片后行HE染色,光镜下观察各组大鼠切牙成釉细胞形态。结果单纯给氟组大鼠。出现牙面粗糙、棕白色相间横纹、白垩色改变,高氟组大鼠的氟斑牙改变较低氟组的改变典型;成釉细胞扭曲变形,正常的高柱状形态丧失,胞内出现空泡等。注射褪黑素组与单纯给氟组的切牙一般状态及成釉细胞形态、排列未见明显差异。结论氟对大鼠切牙的成釉细胞有毒性效应,饮水氟含量高所致氟斑牙症状加重。褪黑素对氟斑牙的发生是否有拮抗作用有待进一步研究。     

不同质量浓度氟对大鼠切牙成釉细胞转化生长因子-β1表达的影响

目的研究不同质量浓度氟对大鼠切牙生长过程中转化生长因子-β1（TGF-β1）表达的影响,探讨氟斑牙的发病机制。方法40只Wistar大鼠随机分为3组,建立氟斑牙动物模型。3组分别为低剂量氟组（F-质量浓度60 mg·L-1,13只）、高剂量氟组（F-质量浓度120 mg·L-1,13只）和对照组（蒸馏水,14只）。10周后取材,采用苏木精-伊红（HE）染色和免疫组织化学染色的方法观察氟对大鼠切牙成釉细胞的形态及TGF-β1表达的影响。结果实验组大鼠切牙均出现典型的氟斑牙症状,牙面出现白垩色改变,釉质表面有横纹。HE染色结果显示成釉细胞形态发生改变,细胞排列紊乱,甚至成灶性堆积,可见空泡性变。免疫组织化学染色结果显示TGF-β1在分泌期和成熟期成釉细胞均为强阳性表达,在星网状层、中间层均为阳性表达,在新形成的釉基质中呈阳性表达。2个实验组TGF-β1的表达强度明显低于对照组（P<0.01）,2个实验组之间的差异无统计学意义（P＞0.05）。结论氟可能通过抑制TGF-β1的表达而干扰了成釉细胞的分化和基质分泌,造成釉质发育障碍。     

Influence of chlorhexidine on in vitro uptake of fluoride in dental enamel

ABSTRACT – Chlorhexidine gluconate and sodium fluoride were found to be compatible in the concentration range of interest in clinical use. Admixture of chlorhexidine to sodium fluoride solutions did not interfere with the fluoride uptake in clinically intact premolars in vitro .     

Influence of (S)-ketoprofen and fluoride on caries in rats

OBJECTIVE: Non-steroidal anti-inflammatory agents (e.g., ketoprofen) used topically appear to be effective in reducing bone loss in the ligature model of periodontitis. Ketoprofen, in common with some food preservatives, e.g., benzoate and sorbate, is a weak acid. Fluoride, too, may behave as a weak acid and, similar to the other agents, may exert antibacterial effects. The purpose of the present study was to determine whether a combination of (S)-ketoprofen, an enantiomer of ketoprofen, alone or in combination with fluoride, would suppress Streptococcus sobrinus populations and reduce the incidence of dental caries in rats. MATERIALS AND METHODS: Toothpastes containing ketoprofen and/or monofluorophosphate were applied to the teeth of six groups of 20 rats twice daily for 5 weeks. RESULTS: Fewest S. sobrinus were found in the group treated with a paste containing 3% (S)-ketoprofen + 0.1% F. This group also displayed the lowest incidence of smooth surface caries of all groups. Severity of sulcal surface caries was also lowest in this group. CONCLUSIONS: Results from this study show that the (S) enantiomer of ketoprofen enhances the caries protective effect of fluoride. It is conceivable that this combination could be effective in combating the two most common maladies of the mouth; periodontal disease and dental caries.     

Micromolar fluoride alters ameloblast lineage cells in vitro

Fluorosed enamel is caused by exposure to fluoride during tooth formation. The objective of this study was to determine whether epithelial ameloblast-lineage cells, derived from the human enamel organ, are directly affected by micromolar concentrations of fluoride. Cells were cultured in the presence of fluoride, and proliferation was measured by BrdU incorporation. The effect of 0, 10, or 20 microM fluoride on apoptosis was determined by the flow cytometry apoptotic index. The effects of fluoride on gene expression were investigated by SuperArray microarray analysis and real-time PCR. Fluoride had a biphasic effect on cell proliferation, with enhanced proliferation at 16 microM, and reduced proliferation at greater than 1 mM F. Flow cytometry showed that both 10 microM and 20 microM NaF significantly increased the apoptotic index of ameloblast-lineage cells. There was no general effect of fluoride on gene expression. These results indicate multiple effects of micromolar fluoride on ameloblast-lineage cells.     

氟离子注入钛表面改性抗菌性能的研究

目的探讨氟离子注入纯钛进行表面改性后,材料表面的化学组成及其对牙龈卟啉单胞菌粘附和形态的影响。方法应用等离子体浸没离子注入装置对钛试件进行氟离子注入,通过X射线光电子能谱分析研究其表面化学组成及各元素百分比;将牙龈卟啉单胞菌接种于氟离子注入前、后的钛样品表面,扫描电镜观察细菌在其表面粘附数量和细胞形态的变化。结果XPS全谱图显示氟离子注入后样品由钛、氧、氟和碳元素组成,且随着氟离子注入时间的延长,材料表面氟元素百分比增高;氟离子注入钛表面改性可影响牙龈卟啉单胞菌的粘附数量和菌体形态。结论氟离子注入可成功的将氟元素引入纯钛表面形成含有金属氟化物的表面改性层,该层可有效地抑制牙龈卟啉单胞菌的粘附并影响其菌体形态,提示其具有抗菌性能。     

Influence of lead on the cariostatic effect of fluoride co-crystallized with sucrose in desalivated rats

OBJECTIVE: Results from previous studies have shown that pre- and perinatal exposure to lead enhances susceptibility of rats to development of dental carieS. A possible explanation for this phenomenon may be that lead complexes with fluoride and renders F insoluble and unable to exert its cariostatic effects. MATERIALS AND METHODS: Thus, to explore this hypothesis, 48 desalivated Sprague-Dawley rats were placed in a König-Höfer programmed feeder and received 17 meals of powdered sucrose daily, and water ad libitum as follows: group (1) plain sucrose and sterile distilled water (SDW); (2) sucrose containing 15 ppm F and SDW; (3) sucrose containing 15 ppm F and 10 ppm Pb water; (4) sucrose containing 15 ppm F and 25 ppm Pb water. RESULTS: The highest smooth-surface, sulcal surface caries and severity scores were observed in group 1.Animals that were exposed to fluoride showed reduced smooth-surface caries and severity scores.S. sobrinus counts did not differ among the groups. CONCLUSION: Lead did not interfere with the protective effect of fluoride in the conditions of the present study.     

Comparative antimicrobial activity of an essential oil and an amine fluoride/stannous fluoride mouthrinse in vitro

Although laboratory studies are not necessarily predictive of clinical activity; they can help to elucidate mechanisms underlying clinical activity when the latter has been established. In a recent clinical study, an essential oil mouthrinse (Listerine Antiseptic) was shown to be significantly more effective than an amine fluoride/stannous fluoride mouthrinse (Meridol) in inhibiting supragingival plaque formation. This paper reports the results of laboratory studies comparing the antimicrobial effectiveness of these 2 mouthrinses using a kill kinetics assay and a plaque biofilm kill assay. In both assays, the essential oil mouthrinse was considerably more effective than the amine fluoride/stannous fluoride mouthrinse. These findings are consistent with the results of the clinical trial and may help to explain the observed differences in clinical activity.