【作者】 白生宾；

【导师】 罗学港； 钟近洁；

【作者基本信息】 中南大学 ， 基础医学， 2012， 博士

【摘要】 第一章慢性氟中毒对大鼠骨相损伤的影响目的：通过慢性染氟,观察氟对大鼠骨相(牙和骨骼)损伤的影响,探讨破骨细胞与氟中毒导致骨相变化的可能关系。方法：本实验采用Wistar大鼠染氟干预(剂量分别是0mg/L、25mg/L、50mg/L、100mg/L、150mg/L)的方法,染氟6个月,分别通过体视显微镜、病理学、X线以及染氟大鼠的氟的负荷量等几个方面检测氟中毒大鼠的特征性指标。结果：①大鼠一般状况的变化,随着染毒时间的延长,染氟第2个月时,100mg/L组和150mg/L组大鼠表现出精神萎靡,食欲减弱；染氟第5个月,50mg/L组大鼠出现上述表现,随着染氟时间的延长,其表现更加明显；实验末期大鼠体重与实验前相比,25mg/L组与空白组比较,大鼠体重值要比空白组低,但统计学显示,差异不具有显著性,(P>0.05)；50mg/L组和100mg/L组与对照组相比较体重增长曲线基本一致,而150mg/L组从第八周开始,体重增长趋势逐渐减弱,与空白组比较,差异具有显著性,(P<0.05)。②大鼠牙齿的变化,大鼠切齿4颗,上齿短,下齿长而尖。对照组大鼠切齿呈均匀的淡黄色,表面光滑有光泽。实验组大鼠个体差异较大,100mg/L、150mg/L组大鼠有的在3个月左右就可出现氟斑牙,轻者牙表面黄、白相间,白垩条纹清晰,尚有一定光泽。随时间的延长,氟斑牙症状逐渐加重,牙齿表面呈无光泽粉笔样白色斑(白垩状)。有的大鼠牙齿表面出现小沟、裂纹、或部分脱落,牙齿呈锯齿状严重缺损。说明染氟剂量对氟斑牙的形成程度有一定的影响,表现出剂量和时间依赖性。③X线征象变化：100mg/L、150mg/L组骨盆的骶髂关节改变最多见。X线征象检查显示骨盆改变出现最早,2个月时少数几例见骨纹模糊,3个月后骨纹模糊、紊乱明显增加骨密度增高,染氟6个月时,染氟150mg/L组部分髓腔密度降低,说明高剂量染氟导致大鼠骨小梁的骨吸收增强,表现出骨质疏松。不同剂量氟干预导致结果不同,主要表现为骨硬化,高剂量表现出骨质疏松。④大鼠股骨组织形态计量参数分析：150mg/L组股骨干骺端平均骨小梁密度(MTPD)及平均骨小梁厚度(MTPT)明显减小,差异有显著性(P<0.05),说明高剂量可以造成骨质疏松的表现。⑤组织病理学改变：150mg/L组：骨板弯曲变形,排列不规则；骨陷窝与骨基质之间的裂隙较多,骨细胞数量减少,胞核皱缩或消失,骨小管细小甚至消失；骨小梁排列紊乱,数量减少,间距变大,宽度变窄,连接呈网状；成骨细胞成层排列,数量增多,破骨细胞数量较其他剂量组明显增多,胞体肥大,细胞核为多个。⑥氟负荷量的变化：染氟6个月后尿液中氟离子随着染氟浓度的升高而呈上升趋势,与对照组比较,差异具有显著性(P<0.05)；血液中氟离子在6个月时,染氟组与对照组比较,氟离子浓度值轻微上升,但统计学比较差异不具有显著性(P>0.05)；大鼠股骨中氟离子浓度值与对照组比较,25mg/L组有轻微上升,但没有统计学意义,而50mg/L、100mg/L、150mg/L三组氟离子上升比较明显,差异具有显著性(P<0.05)；大鼠下切牙的氟离子浓度值与对照组比较,各染氟组都明显升高,差异具有显著性(P<0.05)。结论：染氟后大鼠体内氟负荷量随着时间和剂量基本呈递增趋势；高剂量长时间染氟导致骨质疏松,与破骨细胞的功能活跃有一定的关系。第二章氟对离体培养破骨细胞增殖的影响目的：探讨sRANKL对小鼠RAW264.7细胞的诱导分化及氟对RAW264.7细胞增殖的影响。方法：本实验采用体外培养破骨细胞,以sRANKL诱导RAW264.7细胞进行鉴定,以氟化钠0到160mg/L范围内给出13个剂量组进行染氟,采用噻唑蓝(MTT)法,观察存活细胞数量,并绘制增殖曲线,筛选出与破骨细胞增殖相关的4个剂量组,分别是0、2、10、50mg/L组,用HE染色、甲苯胺蓝染色、TRAP染色以及扫描电镜和透射电镜对破骨细胞的诱导分化及增殖作用进行了形态学和功能方面的鉴定。结果：①诱导前RAW264.7细胞呈星形、圆形或不规则形,细胞核1-2个。诱导后外形仍然呈不规则形,但圆形细胞基本消失,细胞核为多个。TRAP染色阳性细胞为细胞质鲜红色或者淡红色,细胞核2-3个多见,形态呈多突起不规则形。②诱导后倒置显微镜观察与HE染色、甲苯胺蓝染色表明与牛骨磨片共培养发现经过诱导分化后的RAW264.7细胞吸收陷窝明显增多,组间比较差异具有显著性(P<0.05)。③诱导后电镜结果显示,RAW264.7细胞边缘呈指状、刺状或不规则的突起,外形不规则,细胞密度较低的是已分化成熟的破骨细胞,能够形成吸收陷窝。④染氟后通过光镜、电镜及MTT法检测,说明当氟化钠浓度低于2mg/L时,对RAW264.7细胞生长无明显影响；当氟化钠浓度介于2mg/L与10mg/L时,RAW264.7细胞增殖数量明显增加；而当氟化钠浓度高于50mg/L时,RAW264.7细胞增殖受到明显抑制；组间比较差异具有显著性(P<0.05)。结论：明确TRAP染色可以作为破骨细胞一种形态学鉴定方法；低剂量的氟可以促进体外培养小鼠破骨细胞增殖,随剂量增加其促进作用减弱；高剂量氟对破骨细胞增殖有抑制作用。第三章氟对破骨细胞作用的可能分子机制目的：探讨氟对MCM3基因表达和分布的影响以及MCM3对破骨细胞增殖活力的影响。方法：本实验采用体外培养破骨细胞,用免疫荧光技术、Western blot以及半定量RT-PCR的方法,检测不同剂量0、2、10、50mg/L的氟对MCM3mRNA以及蛋白的影响；以及染氟6个月大鼠,用ELISA法检测MCM3在染氟大鼠血清中的变化；通过MTT法检测MCM3对破骨细胞增殖活力的影响。结果：①通过RT-PCR检测比较各组之间MCM3mRNA的表达量,2mg/L组与空白组之间表达量略高,但无统计学意义(P>0.05)；10mg/L组与空白组相比,MCM3mRNA的表达量明显升高(P<0.05)；50mg/L组与空白组相比较MCM3mRNA的表达量明显下降(P<0.05)；各组间比较发现50mg/L组与其他各组具有显著性差异(P<0.05)。②Western-blot结果显示2mg/L、l0mg/L组与空白组相比较,MCM3蛋白表达量略高于空白组(P>0.05),50mg/L组与空白组相比较,MCM3蛋白表达量略低于空白组(P>0.05),2mg/L组与10mg/L组相比较,2mg/L组蛋白表达量略高,但无统计学意义(P>0.05)。③免疫荧光结果显示MCM3蛋白表达0、2、10mg/L各组主要表达在细胞核,有微量在细胞质,表达量依次增强；50mg/L组主要表达在细胞质,细胞核有少量表达,表达强度较弱。④ELISA结果显示高剂量染氟150mg/L组,在染氟6个月时,MCM3的表达量与其他各组比较差异均有统计学意义,P<0.05。⑤体外破骨细胞培养48h内,染氟组在24h～36h,与空白组比较,破骨细胞增殖活力明显提高,有统计学意义,P<0.05；MCM3抗原组在培养24h,增殖活力提高,与空白组比较有统计学意义,P<0.05；当氟与MCM3抗原共同作用时,在破骨细胞培养12h～36h,增殖活力最明显,与空白组比较都有统计学意义,P<0.05；当MCM3抗体作用于破骨细胞,在培养24h～48h时,细胞增殖活力具有下降趋势,与氟+抗原组比较,差异具有统计学意义,P<0.05。说明MCM3抗原相对于抗体在早期就具有刺激破骨细胞增殖的趋势,随着作用时间的延长,MCM3抗原加强了破骨细胞的增殖作用,在染氟晚期,破骨细胞增殖能力也与MCM3有一定的关系。结论：不同剂量的氟对体外培养破骨细胞MCM3的表达量不同,基本表现出2mg/L、10mg/L染氟组促进MCM3的表达,50mg/L染氟组明显抑制了MCM3的表达,从10mg/L氟剂量开始出现抑制作用,随剂量增加促进作用逐渐减弱,50mg/L基本达到毒性反应；高剂量150mg/L氟对在体MCM3表达具有明显增强作用,说明促进破骨细胞增殖,其机制有待进一步研究；MCM3在一定时间内对体外破骨细胞增殖有促进作用,但具体机制需要进一步研究。更多还原

【Abstract】 The chapter I:The effect of chronic fluorosis on bone injury in ratsObjective:To explore possible relationship between osteoclasts and fluorosis bone phase change, based on the observation of fluoride with rat bone (dental and skeletal) damage.Methods:we use in rats exposed to fluoride intervention (dose is respectively Omg/L,25mg/L,50mg/L,100mg/L,150mg/L) method for6months, respectively, at2months,4months and6months for bone density examination, using pathological, radiological and male rats exposed to fluoride fluorine load from several aspects such as the detection of fluorosis rats in effect.Results:①General situation changes of rat, along with the exposure time, in the second months,1OOmg/L and150mg/L rats showed dispirited, appetite diminished; with the extension of time, its performance more obvious. In the fifth months,50mg/L rats appear above the performance of fluorosis. In experimental stage the25mg/L group rat weight compared with blank group, was not significant,(P>0.05).50mg/L group and100mg/L group compared with the control group weight growth curve showed basically the same, while the150mg/L group from the beginning of the eighth week, weight growth trend gradually weakened compared with normal group, the difference was significant (P<0.05)②Rat teeth change, rat teeth were4, upper teeth were short, lower teeth were long sharp. Control rats cutting teeth were uniformly pale yellow, the surface was smooth and shiny. Rat individual differences were large.100mg/L and150mg/L group rats can appear dental fluorosis at about3months, dental surface were light yellow, white, chalky clear stripes and certain gloss. With the extension of time, dental fluorosis symptoms gradually worsened, the tooth surface was dull chalk-like white spot (chalky). Some rat tooth surface was groove, crack, or partially detached, teeth serrated serious defect. fluoride dose on dental fluorosis formation degree have a certain impact, exhibited a dose and time-dependent.③Radiographic changes:100mg/L,150mg/L group pelvis sacroiliac joint changes are common. Radiographic examination revealed pelvic changes appear the earliest, there are a few cases bone pattern fuzzy at2months, after3months bone pattern fuzzy, bone mineral density disorders significantly increased at6months. The part of the medullary cavity density decreased in150mg/L group. It indicated high doses of fluoride in rats trabecular bone absorption enhancement, showed osteoporosis. Different doses of fluoride intervention led to different results, mainly for the bone sclerosis, higher doses showed osteoporosis.④Rat bone histomorphometric parameters analysis:150mg/L femoral metaphysis of mean trabecular bone density (MTPD) and the mean trabecular thickness (MTPT) decreased, there was a significant difference (P<0.05). It indicated high doses can cause osteoporosis performance.⑤Histopathological changes:150mg/L:bone plate bending deformation, the irregular arrangement. The bone lacunae and bone matrix showed the fractured bone, decreased in the number of cells, cell nucleus shrinkage or disappeared, bone tubular became fine and even disappear. Bone trabecular derangement, reduced in number, spacing increased, the width were narrow, connected mesh. Osteoblast arranged many layers, increased in the number. The number of osteoclasts increased significantly than other dose group, the cell body became big, nucleus were multiple.⑥Fluorine load changes:The urine fluoride ions showed increases with the rise in fluoride concentration after6months, compared with the control group, the difference was significant (P<0.05). Blood fluoride ion increased slightly in6months, compared with the control group,but statistically not significant difference (P>0.05). Fluoride ion concentration of rat femur the25mg/L group has increased slightly, but not statistically significant, and the50mg/L,100mg/L,150mg/L three groups of fluoride ion rise apparently, difference significant (P<0.05) compared with the control group. Rat lower incisor fluoride ion concentration were significantly increased in the fluoride exposed group, the difference was significant (P<0.05) compared with the control group. Conclusion:Fluoride load in rats show increased trend with long time and high dose. Fluoride can lead to osteoporosis at a long time and high dose, there is a certain relationship between fluoride and active osteoclast function. The chapter Ⅱ:Affect of fluoride on cultured osteoclasts proliferation in vitroObjective:Explore mouse RAW264.7cells induced differentiation by sRANKL and affect of fluoride on RAW264.7cell proliferation.Methods:we use cultured osteoclasts in vitro induced by sRANKL, RAW264.7cells were identified, using sodium fluoride from0to160mg/L range which were given13dose groups. By the thiazole blue (MTT) method, we observed survival cell number, and the mapping of growth curve, screened and osteoclast proliferation associated4dose groups, respectively was Omg/L,2mg/L, lOmg/L,50mg/L group. HE staining, toluidine blue staining, TRAP staining and scanning electron microscopy and transmission electron microscopy on osteoclast differentiation and proliferation were studied by morphological and functional aspects of the identification.Results:①RAW264.7cells with a star-shaped, rounded or irregularly shaped, nuclei were1-2before induction. The shape remained irregular, but round cells disappeared, the nucleus was more after the induction. TRAP positive staining cytoplasm was bright red or reddish, nuclei was2-3and more, cell shape was irregular.②Resorption lacunae increased significantly difference between two groups was significant after the induction of differentiation of RAW264.7cells (P<0.05)by inverted microscope and HE staining, toluidine blue staining.③Electron microscope results showed RAW264.7cell edge were finger-shaped, spiny or irregular protrusions, irregular shape, low cell density are differentiated mature osteoclasts, capable of forming Howship’s lacunae after the induction④When sodium fluoride concentration is below2mg/L, RAW264.7had no significant effect on cell growth; when the sodium fluoride concentration between2mg/L and10mg/L, RAW264.7cell proliferation in a marked increase in the number; and when the sodium fluoride concentration higher than50mg/L, RAW264.7cell proliferation was inhibited significantly; the difference between groups was significant (P<0.05). Conclusion:We think that TRAP staining can be used for osteoclast a morphological identification method. low fluoride can promote cultured murine osteoclast proliferation, with dose increasing its proliferation effect weakened. high fluoride made osteoclasts proliferation inhibited. The chapter Ⅲ:Possible molecular mechanism of fluoride on osteoclastsObjective:Explore affect of Fluoride on osteoclast MCM3expression and distribution and MCM3on osteoblast proliferationMethods:We use cultured osteoclasts in vitro, immunofluorescence technique, Western blot and semi quantitative RT-PCR detection method, observed MCM3mRNA and protein by different doses of0mg/L,2mg/L,10mg/L,50mg/L fluoride. We detected MCM3in fluoride changes in rat serum when rats ingested fluoride for6months by using ELISA assay. We detected MCM3protein on osteoclast proliferation through the MTT method.Results:①We compared MCM3mRNA expression by the RT-PCR testing.2mg/L group expression was slightly, but not statistically significant (P>0.05) and compared with the control group.10mg/L group MCM3mRNA expression level was significantly elevated (P<0.05) as compared with the blank group.50mg/L group expression of MCM3mRNA decreased (P<0.05) and compared with blank group, each group comparison showed50mg/L group had significant difference (P<0.05)as compared with the other groups.②Western-blot results showed that the2mg/L,10mg/L group expression of MCM3protein was higher than that of the blank group,however (P>0.05).50mg/L group expression of MCM3protein was lower than the blank group,however (P>0.05).2mg/L protein expression was slightly higher, but without statistical significance (P>0.05) between2mg/L and10mg/L group comparison.③Immunofluorescence showed the expression of MCM3protein:0,2,10mg/L were mainly expressed in the nuclei, a trace in the cytoplasm,50mg/L group is mainly expressed in the cytoplasm, nucleus small expression.④ELISA results showed that high doses of fluoride in150mg/L group the expression of MCM3have statistical significance of P<0.05as compared with other groups in the fluoride exposed for6months.⑤There were statistically significant, P<0.05in comparison with blank group in the24h-36h as osteoclast cultured for48hs in vitro. Osteoclast proliferation activity increased significantly. MCM3antigen groups made proliferation activity increase in cultured24hs and it was statistically significant at P<0.05as compared with normal group. Proliferation activity is most obvious in osteoclasts cultured on12h-36h when the fluorine and MCM3antigen acting together, and they were all statistically significant, P<0.05as compared with the blank group. role of, Osteoclasts proliferation activity has decreased when the MCM3antibody group acted for24hs-48hs. it was statistical significance, P<0.05as compared with fluorine+antigen groups. MCM3antigen has the stimulation of osteoclast proliferation trends than antibody in early. along with the prolongation of time, the MCM3antigen strengthen osteoclast proliferation effect of fluoride, osteoclast proliferation and MCM3also have a certain relationship in late.Conclusion:Different doses of sodium fluoride on osteoclastic cells in MCM3expression level is different, basically show2mg/L,10mg/L promotes MCM3expression,50mg/L were significantly inhibited the expression of MCM3, from the lOmg/L dose began inhibition increased with increasing dose, promote gradually decreased,50mg/L reached toxicity reaction. High dose of150mg/L fluoride on MCM3expression has obvious reinforcing effect, that promote osteoclast proliferation, its mechanism needs further research. MCM3at a certain time on osteoclast proliferation activity in vitro, but the exact mechanism needs further study.更多还原