【作者】 李鹏；

【导师】 余擎； 王捍国；

【作者基本信息】 第四军医大学 ， 口腔临床医学， 2010， 硕士

【摘要】 氟化物在口腔医学中有着广泛的应用,在牙本质过敏、防龋、矿化机制和氟斑牙预防研究中扮演重要角色,在生活和临床中被大量使用。已有研究发现高氟暴露的牙本质矿化受到影响,高氟作用下多种细胞的矿化相关因子表达发生变化。牙髓的损伤刺激往往导致局部成牙本质细胞凋亡或坏死,丧失其形成牙本质的功能,同时也观察到受损区牙本质管样结构的修复性牙本质的形成,这说明了成牙本质细胞的凋亡是牙髓损伤修复中的重要现象,在牙髓损伤和修复的中间环节扮演重要角色。又有研究认为氟作用下的细胞膜氯离子通道受到影响,硫的转运增加,有可能与矿化相关蛋白的表达,调控,修饰相互作用。进而推测其影响细胞矿化模式,促进硬组织的损伤修复过程。由于成牙本质细胞在牙本质发育和改建扮演重要角色,能够在人一生之中不断地分泌成牙本质基质,生成修复牙本质。所以研究氟化物诱导下成牙本质细胞凋亡的适宜浓度和时间、牙本质细胞发生凋亡的分子信号通路对于探索氟化物治疗牙本质过敏机制、探寻氟化物调控的硬组织矿化机制有重要的意义。本研究从确定氟诱导的浓度,观察凋亡的发生,以及细胞凋亡的信号通路等不同层次,展开四个实验:实验一:氟对成牙本质细胞的增殖抑制。本实验观察了成牙本质细胞系(odontoblast-lineage cell, OLC)的生物学行为和氟刺激下的细胞形态变化。以不含血清的DMEM培养基孵育12h后,分别换以含NaF 0-6mmol/L浓度的无血清DMEM培养基分别培养12-48h后观察细胞形态变化。继而采用MTT法和台盼兰排斥实验检测氟对OLC细胞的增殖抑制。结果:细胞在氟的作用下增殖分裂缓慢,4mmol浓度作用24小时抑制率可以达到50%以上。推测4mmol浓度作用12-24小时后可能诱导OLC细胞的凋亡出现。实验二:氟诱导的成牙本质细胞系细胞的凋亡为了进一步观察氟诱导成牙本质细胞的凋亡,我们采用流式细胞术、原位末端标记法发现氟化钠能够诱导成牙本质细胞发生凋亡,采用透射电镜技术发现细胞凋亡后的超微结构发生变化,在4mmol/L浓度下,western blot试验中发现了凋亡发生晚期重要的caspase-3蛋白发生了剪切,表明此浓度的氟诱导成牙本质细胞凋亡进入不可逆转的状态。实验三:氟化物通过MAPK途径诱导成牙本质细胞凋亡。为了进一步观察MAPK通路对于成牙本质细胞凋亡的意义,本实验采用western blot技术对JNK、ERK和p38及其磷酸化蛋白进行了检测,发现JNK蛋白随着浓度增加和作用时间延长,磷酸化程度不断增强。ERK的磷酸化过程在时间上表现为短期内升高,降低后又升高的过程.但是随着浓度增加而升高。p38未检测到磷酸化改变。JNK抑制剂SP600125可以有效抑制JNK的磷酸化改变,ERK的抑制剂PD98059可以抑制部分磷酸化过程。这表明JNK信号通路参与了氟诱导的成牙本质细胞凋亡,而ERK信号通路部分参与了此凋亡过程,p38则未参与氟化物诱导的成牙本质细胞凋亡。实验四:线粒体途径对氟化物诱导成牙本质细胞凋亡的影响。为了进一步观察凋亡的另一经典通路,线粒体信号通路对成牙本质细胞凋亡的影响,我们又采用免疫组化法,使用MitoTracker deep red 633线粒体荧光探针标记线粒体,观察Bax在线粒体内的定位情况,结合western blot检测凋亡的线粒体途径中重要的两个分子Bax和Bcl-2的表达情况,发现Bax转位至线粒体并定位于线粒体外膜上,而Bax表达随时间延长而增加,显示凋亡不断进展,Bcl-2作为抗凋亡蛋白,在氟诱导过程中增高以对抗凋亡的发生,48小时后降低可能是凋亡进入不可逆转过程。这些表明了线粒体途径参与了氟诱导的成牙本质细胞凋亡。本研究初步证实了氟化物能够诱导成牙本质细胞系发生凋亡,并进一步发现MAPK通路和线粒体途径都在OLC细胞凋亡中的扮演了重要角色。更多还原

【Abstract】 Fluoride plays an important role in preventing dentin hypersensitivity, remineralization of caries and mechanism of dental fluorosis. It also has been widely used in clinical and oral hygiene. The mineralization of dentin has been found affected by Fluoride exposure related to gene expression changes. For sake of odontoblasts developing function, they keep to converted pulp chamber through human lives, External pressure and temperature stimulations will induced the apoptosis of pulp cells and stimulate migration and differentiation, resulting in reparative dentin.Some studie shows that 3mmol / L of fluoride could not induce apoptosis of odontoblasts . Can fluoride induce apoptosis in odontoblasts ? What is the appropriate time and concentration? Through which molecular pathways did the fluoride-induced apoptosis regulated by? Some studies show that the chloride ion channel was affected by fluoride exposure with an increase of sulfur transfer. It may be associated with the expression, regulation and modification of some mineralization moleculars. what is the relationship between apoptosis-related protein and mineralization-related molecules in odontoblasts ?This research include four experiments:Experiment 1: Cell viability and cell proliferationTo study cell proliferation, OLC cells were exposed to increasing concentrations of NaF at 0-6mmol/L for 12 to 48 hours after in serum-free medium for 12 hours. Cell viability was evaluated using a MTT assay. Results: Incubation of OLC cells with NaF for 12, 24,48 hrs induced a dose-dependent decrease in cell viability .Following exposure to 4 mM NaF for 24 hrs, cell viability was reduced by approximately 50%.Experiment 2: Measurement of apoptosisTo detect the apoptosis induced by fluoride, we use flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to check membrane alteration and DNA fragmentation. we also found the ultrastructural alterations by Scanning electron microscopy. Representative immunoblot obtained with caspase-3 and actin antibodies. Full-length and cleaved forms of caspase-3 were detected .Experiment 3:Involvement of MAPKs in NaF-induced ApoptosisTime-course of NaF-induced accumulation of phosphorylated JNK and phosphorylated ERK in OLC cells. In OLC cells exposed to 4 mM NaF for 24 hrs, treatment with the JNK inhibitor, SP600125 suppressed JNK phosphorylation, while treatment with the ERK inhibitor, PD98059 suppressed ERK phosphorylation partially.Experiment 4: Involvement of mitochondrion in NaF-induced Apoptosis we observe the mitochondrial dynamic morphology by cell stained with MitoTracker Deep Red 633 and Bcl-2 expression increased after flouride exposure.It shows that apoptosis induced by flouride is through the mitochondrial pathway.更多还原