【作者】 蔡偌欣；

【导师】 孙贵范；

【作者基本信息】 中国医科大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 目的氟是人体所必需的微量元素,对人体健康具有双重作用,适量的氟是人体必需的,是构成骨骼和牙齿的重要成分,并且能够促进生长发育和生殖功能。长期大量摄入氟可引起氟中毒,主要表现为氟斑牙和氟骨症。铝是地球上含量最丰富的一种金属,低剂量的铝可刺激骨细胞的增殖和分化,而人体长期摄入铝,在体内蓄积后会引起神经退行性疾病、骨软化和贫血等铝中毒性疾病。研究表明,Al+和F-结合能力比其它60多种金属离子都要强,在酸碱度呈中性条件下,氟化物可与铝离子形成氟化铝,主要形成AlF3和AlF4,AlFx是G蛋白激活因子,与多种酶的激活有关。在氟病区,铝厂、铝矿工人在日常生产活动中,除了接触大量的铝以外,同时还暴露于高氟环境。饮水型氟中毒病区采用铝盐除氟,也造成当地饮用水中氟铝共存现象。很多速食食品中也都同时检出氟、铝含量超标。茶树有强烈富集氟、铝元素的性质,树体内的氟、铝主要富集于成熟叶与老叶中,可高达1,175mg/kg和4,381mg/kg,因此往往导致成熟叶和老叶加工的砖茶中氟、铝含量严重超标,造成饮茶性氟中毒。既往流行病学和动物实验研究发现氟能促进铝在骨骼中蓄积,铝能加重氟中毒的危害,因此饮茶性氟中毒病区的人群骨相损害往往比较严重。所以研究氟铝联合的作用具有重要的现实意义与科研价值。成骨细胞由骨内膜和骨外膜深层以及骨髓的骨祖细胞分化而成。在膜内成骨和软骨内成骨中,充质细胞(MSCs)分化为成骨细胞同时分泌细胞外基质(ECM),随着ECM的钙化,成骨细胞被包埋在骨质中成为骨细胞。其发育和分化可简括为如下几个阶段：间充质干细胞(mesenchymal stem cell)→祖细胞(progenitor cell)→前成骨细胞(preosteoblast)→成熟成骨细胞(mature osteoblast)→骨细胞(osteocyte)。小鼠颅顶前骨细胞亚克隆14 (MC3T3-E1 Subclone 14),是从小鼠的颅顶骨细胞建株的前成骨细胞系,具有ALP活性、Ⅰ型胶原酶合成能力和基质钙化等成骨细胞典型的生物学活性。本研究利用MC3T3-E1细胞系研究不同剂量氟、铝和氟铝联合染毒后对MC3T3-E1细胞的增殖和分化能力的影响,并探讨氟铝联合染毒后对核心结合因子-al(Runx2)、Osterix、骨保护素(Osteoprotegerin, OPG)、细胞核因子κB受体活化因子配体(receptor activator of nuclear factorκB ligand, RANKL)的影响,为进一步阐明地方性氟中毒骨相损害机制的研究提供实验依据。方法一、实验方法1、细胞培养小鼠颅顶骨前成骨细胞亚克隆14(MC3T3-E subclone 14)细胞常规培养于a-MEM培养基(含10%胎牛血清,1×105U／L青、链霉素),5%CO2、37℃条件下培养。2、配制染毒液配制100mM NaF和10mM AlCl3的储备液。在染毒前稀释为工作液,分别为500μM和50μM,为最终染毒剂量的10倍。联合染毒液根据Chabre的方法,以NaF1 mM与AlCl3100 mM 1：1混合,现用现配。3、细胞增殖活力能力检测采用CCK法。实验分组为：对照组、单纯染氟组、单纯染铝组和氟铝联合染毒组。用酶标仪在450 nm处测定各孔的光密度(OD)值。每个剂量组设5个平行样。4、细胞周期检测采用细胞周期试剂盒检测细胞周期的变化。实验分组为：对照组、单纯染氟组、单纯染铝组和氟铝联合染毒组。用FAC Scan流式细胞仪,488 nm处检测红色荧光5、ALP活性测定本实验ALP活性的测定采用ALP test kit。细胞ALP活性的测定根据以下原理：磷酸对硝基苯酚在ALP存在时,能转化为对硝基苯酚和磷酸盐。实验分组为：对照组、单纯染氟组、单纯染铝组和氟铝联合染毒组。6、细胞总RNA的提取采用常规RNA提取法,以Trizol来提取MC3T3-E1细胞中的RNA。实验分组为：对照组、单纯染氟组、单纯染铝组和氟铝联合染毒组。7、引物合成从GenBank下载其cDNA序列,根据文献和专用设计软件Primer 5设计并合成相应的mRNA引物；检索小鼠看家基因(β-actin)、OPG、RANKL、Osterix、Runx2 cDNA序列,由上海生工生物公司设计并合成。8、RT-PCR取细胞总RNA 3μl,检测RNA浓度。用前调整浓度至0.5μg／μl。RT总反应体积为5μl。PCR总反应体积为20μl,具体步骤如下：cDNA 5μl,5×PCR Buffer 5μl,上下游引物(10μM)各0.25μl,Taq酶0.125μl,补水14.375μl,混匀于0.5 mlEP管中。二、统计分析用SPSS 16.0统计软件进行统计分析,组与组之间用两组独立样本t-test;组间比较采用单因素方差分析(ANOVA)。结果1、MC3T3-E1细胞形态观察未贴壁的MC3T3-E1细胞呈圆形,经培养24 h后,在倒置显微镜下可见细胞贴壁生长,细胞呈三角形、纺锤形和多角形等多种形态。形态相似的细胞成堆出现,胞浆伸展,并伸出2-4个突起,细胞核明显,核仁清晰(见图1)。3～4 d,细胞呈指数增长,细胞体积增大,胞浆丰富。6-7 d,细胞融合呈铺石状排列(见图2)。2、氟对MC3T3-E1细胞增殖能力的影响由表1可见,10-9～10-4 M F-作用24～72 h后对MC3T3-E1细胞无促进增殖作用,1 mM F-显著抑制MC3T3-E1细胞增殖(p<0.01)。3、氟、铝及氟铝联合对MC3T3-E1细胞增殖的影响由表2可见,与对照组相比,氟(50μM)或铝(5μM)染毒72 h后对MC3T3-E1细胞无促进增殖作用；氟铝联合染毒(F 50μM+Al5μM)显著促进MC3T3-E1细胞的增殖(p<0.01)。与对照组相比,氟铝联合作用后,增殖率增加了9.2%。4、氟、铝及氟铝联合对MC3T3-E1细胞周期的影响从表3可以看出,与对照组相比,氟(50μM)、铝(5μM)对细胞周期影响不明显；而氟铝联合染毒72 h后,G2／M期细胞显著增多(p<0.05),PI以及DNA相对含量显著增高(p<0.05)5、氟、铝及氟铝联合对MC3T3-E1细胞ALP活性的影响由表4可以看出,对照组相比,氟(50μM)或铝(5μM)矿化液染毒5 d后,MC3T3-E1细胞ALP表达量无明显变化；氟铝联合染毒(F 50μM+Al5μM)显著促进MC3T3-E1细胞的ALP表达(p<0.05),刺激MC3T3-E1细胞分化。6、RT-PCR结果6.1染毒72h氟、铝及氟铝联合对Runx2、Osterix mRNA表达的影响从图7、8可以看出,染毒72 h后,与对照组相比,单纯染氟组(50μM)或单纯染铝组(5μM) Runx2、Osterix mRNA表达无明显变化；氟铝联合染毒组(F50μM+Al5μM)显著促进Runx2 mRNA的表达(p<0.05)；氟铝联合染毒组(F50μM+Al 5μM) Osterix mRNA表达显著增高,有统计学差异(p<0.01)。6.2染毒72h氟、铝及氟铝联合对OPG、RANKL mRNA表达的影响从图10、11、12可以看出,染毒72 h后,与对照组相比,单纯染氟组(50μM)或单纯染铝组(5μM) OPG mRNA的表达无明显变化；氟铝联合染毒组(F 50μM+Al5μM)显著促进OPG mRNA的表达(p<0.05)。各组的RANKL mRNA表达无显著性差异。与对照组相比,氟铝联合染毒组(F 50μM+Al 5μM) RANKL/OPG比值降低,有统计学差异(p<0.05)。结论1、氟铝联合能够显著提高对MC3T3-E1细胞的增殖,并且能显著提高M期细胞的含量,促进成骨细胞由S期向M期转化,促进成骨细胞增殖。2、氟铝联合能增加MC3T3-E1细胞ALP活性,促进成骨细胞分化。3、氟铝联合能够显著提高MC3T3-E1细胞Runx2、Osterix mRNA的表达量,促进成骨细胞形成。氟铝联合能够显著促进OPG mRNA的表达,下调RANKL/OPG比值,使骨重建失衡,从而起到抑制骨吸收、促进骨形成的作用。更多还原

【Abstract】 ObjectiveFluoride is an essential trace element for human. An appropriate intake of fluoride increase bone density and promote teeth growth, promote the growth and generation functions as well. However, chronic excessive intake of fluoride may cause fluorosis. Aluminum is the most abundant on earth, an appropriate intake of aluminum can stimulate bone cell proliferation and differentiation. However, excessive intake of aluminum could accumulate in the body, might cause senile dementia, osteomalacia, anemia and other disease. Studies have shown that, the capacity of Al+ binding with F-in neutral pH condition is better than other 60 kinds of metal ions. Fluoride in a complex with aluminum (fluoroaluminate, most likely AlF4-) binds to G proteins in vitro and activates G protein-mediated intracellular signaling pathways. In the fluorosis area, aluminum workers not only exposed to large number of aluminum, but also exposed to high fluoride. In water fluorosis areas, people use aluminum to remove fluoride, caused the coexistence of aluminum and fluoride in drinking water. A lot of instant foods are also detected aluminum fluoride content exceeded. Tea has a strong accumulation capacity of fluoride and aluminum, usually fluoride and aluminum are enriched in mature leaves and old leaves, which up to 1175mg/kg and 4381mg/kg, resulted in the tea drinking fluorosis. The epidemiological and animal experimental study found that fluoride can promote the accumulation of aluminum in bones, aluminum can promote the fluoride poisoning. Hence in the tea-drinking fluorosis ward, damage to the patients are often more serious. Therefore, the study of aluminum fluoride has an important practical significance and research value.The osteoblast cell was derived from mesenchymal stem cell. The development may experience several stages as follows:mesenchymal stem cell→progenitor cell→preosteoblast→mature osteoblast→osteocyte。We use MC3T3-E1 cells to explore the effect of different concentrations of fluoride, aluminum and aluminiunofluoride on proliferation and differentiation, investigate the mechanism of endemic fluorosis and provide experimental data.Methods1. Cell CultureMC3T3-E1 cells were cultured in aMEM conventional medium containing 10% fetal bovine serum,1×105U/L penicillin, streptomycin,5% CO2, cultured under the conditions of 37℃.2. MaterialsThe stock solutions of NaF and AlCl3 (100mM和10mM, respectively), as well as working solutions at 10 times higher concentration than final, were prepared in water and mixed only before the experiment.3. Cell proliferation dynamic detectedThe cell proliferation was measured by CCK method. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group. OD was measured with the microplate reader at 450 nm.4. Cell cycleThe change of cell cycle was measured by flow cytometry. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.5. ALP activity assayThis experiment was determined by ALP activity test kit. Determination of cellular ALP activity based on the following principle:phosphorylation of p-nitrophenol in the presence of ALP can be transformed into p-nitrophenol and phosphate. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.6. Extraction of total cellular RNAWe used Trizol to extract the MC3T3-E1 cell RNA. Experimental groups as follows:control group, fluoride group, aluminum group, aluminiunofluoride group.7. Primer synthesisAccording to documents the cDNA sequences download from the GeneBank, and designed by software Primer 5. Designed and synthesized. The mouse OPQ RANKL, housekeeping gene (β-actin), Osterix, Runx2 cDNA sequence by the Shanghai Public Health Biology company designed and synthesized.8. RT-PCRTake total cellular RNA 3μl, detection of RNA concentration. Before using adjust the concentration to 0.5μg/μl. RT total reaction volume of 5μl. The total reaction volume of 20μl, concrete steps are as follows:cDNA5μl,5×PCR Buffer 5μl, upstream and downstream primers (10μM) of 0.25μl, Taq enzyme 0.125μl, replenishment 14.375μl, mixing in 0.5ml EP tubes.Statistical analysisUsing SPSS 16.0 statistical software for statistical analysis, group and between groups using two independent samples t-test; groups compared using single factor analysis of variance (ANOVA),22 compared the use of LSD-t test.Results1. MC3T3-E1 cell morphologyThe suspensioned MC3T3-E1 cells were round. After cultured 24 h, in an inverted microscope could see the cell had been adherent, in the form of triangular, spindle-shaped, and a variety of polygonal forms. Piles of morphologically similar cells appeared cytoplasmic extension, with 2 to 4 and protruding nuclei obvious nucleolus definition (see as Figurel). After 3-4 d, cells in exponential growth, cell volume increased, rich in cytoplasm. After 6-7 d, cell fusion was stone pavement-like arrangement (see as Figure 2).2. Fluoride on the MC3T3-E1 cell proliferationTable 1 shows that after 24-72 h, compared with the control group, fluoride alone did not promote MC3T3-E1 cells proliferation.3. Fluoride, aluminum and aluminiunofluoride on cell proliferationTable 2 shows that after 72 h, compared with the control group, fluoride (50μM) or aluminum (5μM), did not promote MC3T3-E1 cells proliferation; Aluminiunofluoride stimulated proliferation in MC3T3-E1 cells (p<0.01). Compared with the control group, aluminiunofluoride group the proliferation rate had increased by 9.2%.4. cell cycleAs seen from Table 3, compared with control group, fluoride (50μM), aluminum (5μM) had no obvious effect on cell cycle. Aluminiunofluoride significantly induced increase of G2/M phase, PI (proliferation index) and DNA relative content.5. Fluoride, aluminum and aluminiunofluoride on the ALP activity.As can be seen from table 3 after 5 d mineralization fluid exposure, compared with the control group, F (50μM) or aluminum (5μM) group, ALP expression had no significant change; aluminiunofluoride significantly promoted ALP expression (p<0.05), stimulated MC3T3-E1 cell differentiation.6. RT-PCR results6.1. Fluoride, aluminum and aluminiunofluoride on the OPG, RANKL mRNA expression.As can be seen from graph7、8 after 72 h exposure, compared with the control group, aluminiunofluoride group (F 50μM+Al 5μM) significantly promoted the OPG mRNA expression (p<0.05). The RANKL mRNA did not have difference in expression. Compared with the control group, the aluminiunofluoride group (F 50μM+Al 5μmol /L), RANKL/OPG ratio decreased, there is significant difference (p<0.05).6.2. Fluoride, aluminum and aluminiunofluoride on the Runx2, Osterix mRNA expression.As can be seen from graph 10、11、12, after 72 h exposure, compared with the control group, simple fluoride group or pure aluminum group had no significant change in the expression of Runx2, Osterix mRNA; aluminiunofluoride group significantly enhanced Runx2 mRNA expression (p<0.05); in the aluminiunofluoride group Osterix mRNA expression was significantly increased (p<0.01).Conclusions1. Fluoride alone did not elicit any proliferation in osteoblast, while the aluminiunofluoride could significantly improve MC3T3-E1 cells proliferation and the content of M-phase cell, modulate MC3T3-E1 cells from S phase to the M phase transformation.2. Aluminiunofluoride could increase the activity of ALP. Aluminiunofluoride could significantly increase MC3T3-E1 cells differentiation capacity.3. Aluminiunofluoride can significantly improve the expression of Runx2 and osterix mRNA, stimulate MC3T3-E1 cells differentiation and bone formation. Aluminiunofluoride could significantly enhance the expression of OPG mRNA and reduce the RANKL/OPG ratio, balance the RANKL/OPG in reconstruction.Innovative self-evaluationWe study the initiation links of fluorosis in the perspective of aluminiunofluoride, study fluoride, aluminum and aluminiunofluoride on the MC3T3-E1 cells proliferation, differentiation, and mineralization capability. At present, bone reconstruction is a hot issue. According to OPG/RANKL ratio change and Runx2、Osterix mRNA expression we explore how fluoride modulates bone formation and absorption relationship in the process of signal transduction.更多还原