Excess fluoride interferes with chloride-channel-dependent endocytosis in ameloblasts

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF). Both CF and dental fluorosis result in protein retention in mature enamel. We hypothesized that excess fluoride might cause protein retention by interfering with CFTR function, resulting in abnormal expression of proteases and pathological endocytosis. Millimolar concentrations of fluoride reduced uptake of Emdogain, an enamel matrix derivative, in ameloblast-like PABSo-E cells, while stimulating an acidic intracellular environment at the same time. When CFTR function was inhibited by either an siRNA or a chloride channel inhibitor, CFTRinh-172, fluoride's effect on Emdogain uptake was partially blocked. Treatment of cells with CFTR siRNA down-regulated expression of proteases MMP20 and KLK4 and increased intracellular pH. We conclude that excess fluoride inhibits endocytic activity of ameloblasts through the CFTR chloride channel or other chloride channels. The intracellular pH might be the key mechanism by which abnormal proteolytic activity and defective endocytosis cause the residual protein observed in enamel of patients with CF and dental fluorosis.     

过量氟诱导成釉细胞钙超载及细胞凋亡的实验研究

目的 研究过量氟对体外培养大鼠成釉细胞内钙超载及细胞凋亡的影响。方法 取大鼠成釉细胞系HAT-7细胞,分别加入不同浓度（0、0.4、0.8、1.6、3.2、6.4 mmol·L^-1）的氟化钠培养液,培养48 h后,采用Cell Counting Kit 8（CCK-8）试剂盒检测各组细胞的活性,流式细胞术分析氟对细胞凋亡的影响,激光扫描共聚焦显微镜、Western blot试验和实时荧光定量聚合酶链反应技术检测过量氟诱导大鼠成釉细胞内Ca²⁺浓度和钙网蛋白表达的变化。结果 氟化钠浓度高于1.6 mmol·L^-1时,可抑制成釉细胞的活性,成釉细胞内Ca²⁺浓度升高,钙网蛋白表达上调,细胞早期凋亡数量增加,并且随着浓度的增加,细胞凋亡的数量也随之增加。结论 过量氟可引起成釉细胞内钙超载,诱导成釉细胞凋亡。     

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。     

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。     

短期高浓度氟对小鼠磨牙成釉细胞形态及骨形成蛋白-4表达的影响

目的:通过研究短期高浓度氟对小鼠磨牙成釉细胞形态及骨形成蛋白-4(bone morphogenetic protein-4,BMP-4)表达的影响,探讨氟牙症形成的可能机制。方法:选择4d龄的ICR小鼠共32只,随机分为2组,每组16只,再分实验动物和对照动物各半。实验动物单次腹腔注射剂量分别为10mg/kg和20mg/kg的NaF,对照动物单次腹腔注射等剂量的NaCl,注射量均为10μl/g。24h后处死动物。采用HE染色、免疫组化染色观察高浓度氟对小鼠磨牙不同分化阶段成釉细胞形态及BMP-4的表达,采用SPSS13.0软件对数据进行分析。结果:分泌早、晚期和过渡期的成釉细胞对短期暴露高浓度F-敏感,实验动物分泌晚期和过渡期的成釉细胞中BMP-4的表达明显弱于对照动物,差异有统计学意义(P<0.01),而成熟期成釉细胞未见明显变化。结论:短期高浓度氟可抑制BMP-4在分泌晚期和过渡期成釉细胞中的表达,影响釉质发育。     

Utilization of ( 3 H)-serine by ameloblasts of rats receiving sub-mottling doses of fluoride

Five-day-old Wistar rats were given three intraperitoneal injections at 2-hourly intervals of a solution of sodium fluoride in 0.9 per cent sodium chloride. Three fluoride levels were used: a mottling dose of 3 mgF/kg body weight; and two sub-mottling doses, 0.05 mg and 0.01 mgF/kg body weight. Thirty minutes after the last injection, each rat received 5 μCi/g body weight of [³H]-serine. The design allowed for within-litter comparisons of treatments to be made. Rats were killed 1 hr and 20 hr after the injection of the label, and the tissues were processed for light microscope autoradiography.     

Short exposure to high levels of fluoride induces stage-dependent structural changes in ameloblasts and enamel mineralization

We tested the hypothesis that the sensitivity of forming dental enamel to fluoride (F^–) is ameloblast developmental stage-dependent and that enamel mineralization disturbances at the surface of fluorotic enamel are caused by damage to late-secretory- and transitional-stage ameloblasts. Four-day-old hamsters received a single intraperitoneal dose of 2.5–20 mg NaF/kg body weight and were examined, 24 h later, by histology and histochemistry. A single dose of ≥ 5 mg of NaF/kg induced the formation of a hyper- followed by a hypomineralized band in the secretory enamel, without changing the ameloblast structure. At 10 mg of NaF/kg, cystic lesions became apparent under isolated populations of distorted late-secretory- and transitional-stage ameloblasts. Staining with von Kossa stain showed that the enamel under these lesions was hypermineralized. At 20 mg of NaF/kg, cystic lesions containing necrotic cells were also found in the early stages of secretory amelogenesis and were also accompanied with hypermineralization of the enamel surface. We concluded that the sensitivity to F^– is ameloblast developmental stage-dependent. Groups of transitional ameloblasts are most sensitive, followed by those at early secretory stages. These data suggest that a F-induced increase in cell death in the transitional-stage ameloblasts accompanies the formation of cystic lesions, which may explain the formation of enamel pits seen clinically in erupted teeth.     

高氟对成釉细胞内氯离子浓度及pH值影响的研究

目的：检测高氟对成釉细胞内氯离子浓度以及pH值的影响。方法：2mmol／LNaF处理成釉细胞样细胞系LS8 30min,采用新型氯离子荧光探针MQAE以及LysoSensorTMDND-167分别显示细胞内氯离子浓度和pH值的变化;利用活细胞工作站实时监测加氟30min后细胞内氯离子浓度和pH值的变化。结果：MQAE呈现绿色荧光,其辉度值与细胞内的氯离子呈反比;加氟细胞30min后,实验组细胞平均绿色荧光强度较对照组增高;活细胞检测显示,对照组蓝色荧光和绿色荧光均少许下降,而实验组细胞双染后两种荧光均有所增强。结论：氟离子能影响细胞内氯离子浓度从而引起细胞内DH佰的降低而导致细胞的酸化。     

枸杞多糖和原花青素对小鼠成釉细胞DNA损伤作用的研究

目的：探讨枸杞多糖和原花青素对小鼠成釉细胞DNA损伤的保护作用。方法：小鼠成釉细胞以100．00、200．00、400．00mg／L枸杞多糖作为枸杞多糖作用组,以10．00、25．00、50．00mg／L原花青素作为原花青素作用组。细胞染毒24h后,采用单细胞凝胶电泳技术（SCGE）检测DNA单链断裂情况。结果：与对照组比较,100．00、200．00和400．00mg／L枸杞多糖作用组小鼠成釉细胞尾长、Olive尾距和尾长／头长值均明显下降,差异具有统计学意义（P〈0．05）。100．00、400．00mg／L枸杞多糖作用组尾DNA％较对照组明显下降（P〈0．05）。与对照组比较,10．0HD、25．00、50．00mg／L原花青素作用组小鼠成釉细胞DNA损伤的尾长、Olive尾矩、尾DNA％、尾长／头长值均较对照组均显著性下降,差异具有统计学意义∽〈0．05）。以Olive尾矩为观察指标,100．00mg／L枸杞多糖作用组的Olive尾矩明显低于200．00mg／L和400．00mg／L枸杞多糖作用组（尸〈0．05）,10．00mg／L原花青素作用组的Olive尾矩明显低于25．00mg／L和50．00mg／L原花青素作用组（尸〈0．05）。结论：100．00～400．00mg／L枸杞多糖和10．00～50．00mg／L原花青素对小鼠成釉细胞DNA损伤具有较好的保护作用,以100．00mg／L枸杞多糖、10．00mg／L原花青素的保护作用相对较好。     

EGF/EGFR/ErbB2调控成釉细胞MMP-20基因表达的研究

目的:研究EGF/EGFR/ErbB2信号通路对小鼠成釉细胞中基质金属蛋白酶-20(matrix metalloproteinase-20,MMP-20)基因表达的调控作用.方法:用定时定量RT-PCR、双荧光素酶报告基因检测系统分析EGF/EGFR/ErbB2调控MMP-20基因的表达、EGF调控MMP-20启动子的主要区域、MAPK信号通路阻断剂对EGF调控MMP-20转录活性的影响、转录因子Jun家族介导EGF/ErbB2对MMP-20转录活性的调控;用基因定点突变和双荧光素酶基因检测报告系统分析EGF/ErbB2、C-Jun对MMP-20基因启动子转录活性的影响.结果:成釉细胞经20 ng/mL EGF刺激后,MMP-20的基因表达上调(P＜0.05),MMP-20启动子在(-157～+23)之前的区域转录活性增强(P ＜0.05);EGFR、ErbB2、ERK阻断剂均能抑制EGF对MMP-20基因表达的调控(P＜0.05);转录因子C-Jun介导EGFR/ErbB2上调MMP-20基因的表达(P＜0.05);突变MMP-20启动子的AP1结合位点后,MMP-20基因表达的水平下降(P＜0.05),同时EGF/ErbB2也失去上调MMP-20基因表达水平的作用.结论:在成釉细胞中EGF通过EGFR/ErbB2 ～ ERK～C-Jun信号通路对MMP-20基因的表达起到调控作用.     

Mandible fracture severity may be increased by alcohol and interpersonal violence

Background: Alcohol as a cofactor in interpersonal violence (IPV) has been established by studies from a number of countries. This study aimed to determine if alcohol was a cofactor in the incidence or severity of mandible fracture. Methods: A prospective study of mandible fracture patients presenting for oral maxillofacial review over 16 months was completed. Injury severity was assessed utilizing the Mandible Injury Severity Score (MISS). Results: A total of 252 facial trauma cases presented to our tertiary referral centre, 83 with fractures of the mandible. The majority of presentations were secondary to IPV (n = 54, 65.06%), 49 (90.74%) of these cases involved alcohol. Overall, alcohol was involved in 63.85% of cases (n = 53). The relative risk of requiring surgical intervention with alcohol involvement was 2.68 (CI = 1.11–9.47). Alcohol significantly increased facial fracture severity for MISS: alcohol (n = 53) 13.07 ± 5.01, no alcohol (n = 30) 11.03 ± 4.87 (p < 0.05). IPV also increased facial fracture severity for MISS: IPV (n = 54) 13.09 ± 4.90, non‐IPV (n = 29) 11.00 ± 4.81 (p < 0.05). The angle of the mandible was most commonly fractured (40.5% of cases). Conclusions: Mandible fracture patients, whose injury is a result of IPV, have more severe fractures and a higher likelihood of requiring surgery if alcohol is involved. This correlates to a higher surgical workload, economic and social burden to the community. Primary alcohol and IPV prevention strategies will play an important role in reducing mandible fracture.     

IL-1 genotype may be a contributing factor to disease progression

J Evid Base Dent Pract 2002;2:237-8     

There may be a new association between IL-1 genotype and adult periodontitis

J Evid Base Dent Pract 2002;2:140-42     

In vitro fluoride uptake, distribution and retention by human enamel after 1- and 24-hour application of various topical fluoride agents

The in vitro fluoride acquisition by human enamel after a 1-hour and 24-hour application of APF, Duraphat (a resin varnish) or Fluor Protector (a polyurethane varnish) and subjection to various procedures was determined. Fluoride acquisition was the greatest in teeth treated with Fluor Protector and the least in APF-treated teeth. Fluoride uptake and distribution were increased by prolonging the contact time between the varnishes and enamel, and fluoride retention was decreased after subsequent exposure to synthetic saliva.