氟对破骨细胞增殖的影响及可能的分子机制

发布时间：2018-01-10 13:00

本文关键词：氟对破骨细胞增殖的影响及可能的分子机制　出处：《中南大学》2012年博士论文　论文类型：学位论文

【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,(P0.05)；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,(P0.05)。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(P0.05),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(P0.05)；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(P0.05)；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(P0.05)；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(P0.05)。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(P0.05)。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(P0.05)。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(P0.05)；10mg/L组与空白组相比,MCM3mRNA的表达量明显升高(P0.05)；50mg/L组与空白组相比较MCM3mRNA的表达量明显下降(P0.05)；各组间比较发现50mg/L组与其他各组具有显著性差异(P0.05)。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(P0.05),50mg/L组与空白组相比较,MCM3蛋白表达量略低于空白组(P0.05),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(P0.05)。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,P0.05。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,P0.05；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,P0.05；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,P0.05；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,P0.05。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。
[Abstract]:The effect of chronic fluorosis in the first chapter on the injury of bone in rats
Objective: To observe the effect of fluorine on the injury of bone phase (tooth and bone) in rats by chronic fluorine dye, and to explore the possible relationship between osteoclast and fluorosis.
Methods: the Wistar rats were exposed to fluoride intervention (doses were 0mg/L, 25mg/L, 50mg/L, 100mg/L, 150mg/L) method of fluoride for 6 months, respectively, through the microscope, pathology, X-ray and several characteristic indexes in rats exposed to fluorine fluorine load on detection of fluorosis rats.
Results: the changes of general condition of rats, with the extension of exposure time, the fluoride at second months, 100mg/L group and 150mg/L group rats showed listlessness, decreased appetite; fluoride fifth months, rats in the 50mg/L group appear in the show, with extended exposure to fluoride, which is more obvious compared with the experimental rats; body weight before the end of the experiment, 25mg/L group compared with the control group, the rats weight value lower than the control group, but the statistics showed that the difference was not significant, (P0.05); 50mg/L group and 100mg/L group compared with the control group, the weight gain of Qu Xianji consistent, while the 150mg/L group from the beginning of the eighth weeks, weight increased gradually, compared with the control group, the difference was significant (P0.05). The changes of rat teeth, tooth rat 4 teeth, teeth short, long and sharp teeth. The rats in control group were evenly cutting pale yellow, shiny surface. The experimental group In individual differences, 100mg/L, 150mg/L group of rats in 3 months or so can appear dental fluorosis, tooth surface light yellow, white chalk stripe is clear, there is a certain luster. With the extension of time, dental fluorosis symptoms gradually worsened, the tooth surface was dull chalk like white spots (Bai Ezhuang). The minor groove, there some rat tooth surface crack, or shedding of jagged teeth defect. That has a certain impact on the formation of fluoride concentration degree of dental fluorosis, showed a dose and time dependent manner. The X-ray signs of change: 100mg/L, 150mg/L group of pelvic sacroiliac joint change is the most common. X-ray examination showed pelvic changes the earliest, 2 months, a few cases of bone lines blurred, 3 months after the bone pattern fuzzy disorder obviously increase the bone density increased, fluoride at 6 months, the fluoride group 150mg/L part of the medullary cavity density decreased, indicating a high dose fluoride Guide Absorption induced rat trabecular bone increased, showed osteoporosis. Different doses of fluoride intervention leads to different results, mainly for bone sclerosis, high dose showed osteoporosis. Analysis of the rat femur histomorphometric parameters: group 150mg/L femoral metaphyseal mean trabecular bone density (MTPD) and the average bone Liang Houdu (MTPT) was significantly reduced, there were significant differences (P0.05), high dose can cause osteoporosis. The pathological changes: 150mg/L group: the bending deformation of the bone plate, irregular arrangement; fissure more between the lacunae and bone matrix, bone cells decreased, nucleus shrinkage or disappearance fine, canaliculi or even disappear; reduce the number of trabecular derangement, spacing becomes larger, width, connected with reticulate; osteoblast layers, the number increased, the number of osteoclasts than other dose groups significantly increased, the cell body hypertrophy, fine The nucleus is multiple. The change of fluorine load: fluoride after 6 months in the urine fluoride with increasing fluoride concentration increased, compared with the control group, the difference was significant (P0.05); the blood fluoride in 6 months, fluoride group compared with the control group and the fluorine ion concentration value increased slightly, but the difference was not significant (P0.05); compared with the control group of fluorine ion concentration in the rat femur, 25mg/L group has increased slightly, but not statistically significant, while 50mg/L, 100mg/L, 150mg/L three groups of fluorine ion rise is more obvious, the difference was significant (P0.05); rat incisor fluoride concentration values compared with the control group, the fluoride group were significantly increased, the difference was significant (P0.05).
Conclusion: fluoride load in rats increases basically with time and dose after exposure to fluoride. High dose of fluoride for a long time can lead to osteoporosis, which is related to the functional activity of osteoclasts.
The effect of fluorine on the proliferation of vitro cultured osteoclasts in the second chapter
Objective: To investigate the effect of sRANKL on the induced differentiation of mouse RAW264.7 cells and the effect of fluorine on the proliferation of RAW264.7 cells.
Methods: this experiment used cultured osteoclasts in vitro by sRANKL induced RAW264.7 cells were identified by sodium fluoride in the range of 0 to 160mg/L are given 13 doses of fluoride, methyl thiazolyl tetrazolium (MTT) method, observe the number of surviving cells, and draw the growth curve, screened 4 dose groups associated with osteoclast cell proliferation, respectively 0,2,10,50mg/L group, with HE staining, toluidine blue staining, differentiation and proliferation induced by TRAP staining and scanning electron microscopy and transmission electron microscopy of osteoclasts were identified by morphological and functional aspects.
Results: before induction of RAW264.7 cells with a star shaped, round or irregular shape, nuclear 1-2. After induction of appearance is still irregular, but round cells disappeared, the nucleus for multiple.TRAP staining positive cells were bright red or pale red, a rare form of nuclear 2-3, showed irregular projections after induction was observed with HE. The inverted microscope staining, toluidine blue staining showed that co cultured with bovine bone slices found after induction of differentiation of RAW264.7 cells after resorption increased significantly, the difference between groups was significant (P0.05). The results showed induction after RAW264.7 cells at the edge with a finger, spiny or irregular projection, irregular shape, cell density is lower in the differentiation of mature osteoclasts, can be formed by light microscopy. The resorption of fluoride detection, electron microscope and MTT method, when the concentration of sodium fluoride is less than 2mg/L When the concentration of sodium fluoride was between 2mg/L and 10mg/L, the proliferation of RAW264.7 cells increased significantly. When the concentration of sodium fluoride was higher than 50mg/L, the proliferation of RAW264.7 cells was significantly inhibited, and the difference between groups was significant (P0.05). When the concentration of sodium fluoride was between 10mg/L and 50mg/L, the proliferation of RAW264.7 cells was significantly inhibited.
Conclusion: TRAP staining can be used as a morphological identification method for osteoclasts. Low dose fluoride can promote the proliferation of osteoclasts in vitro, and its promotion effect decreases with the increase of dose. High dose fluoride inhibits the proliferation of osteoclasts.
The possible molecular mechanism of fluorine effect on osteoclast in the third chapter
Objective: To investigate the effect of fluorine on the expression and distribution of MCM3 gene and the effect of MCM3 on the proliferation of osteoclast.
Methods: this experiment used cultured osteoclasts in vitro by immunofluorescence, Western blot and semi quantitative RT-PCR method, detection of different doses of 0,2,10,50mg/L and the effects of fluoride on MCM3mRNA protein; and fluoride 6 months rats were detected by ELISA MCM3 staining in the changes of serum fluoride in rats by impact; detection of MCM3 MTT method to break the proliferation activity of osteoblasts.
Results: the expression of MCM3mRNA was detected by RT-PCR between the groups were compared between 2mg/L group and control group, the expression is slightly higher, but there was no statistical significance (P0.05); 10mg/L group compared with the control group, the expression of MCM3mRNA was significantly increased (P0.05); 50mg/L group compared with the control group the expression of MCM3mRNA was significantly decreased (P0.05); comparison between groups showed that 50mg/L group had a significant difference with other groups (P0.05). The Western-blot results showed that 2mg/L, l0mg/L group compared with the control group, MCM3 protein expression was slightly higher than that of the control group (P0.05), 50mg/L group compared with the control group, the expression of MCM3 protein was slightly lower than that of the control group (P0.05), 2mg/L group and 10mg/L group compared to 2mg/L group, the protein expression is slightly higher, but there was no statistical significance (P0.05). The immunofluorescence results showed that 0,2,10mg/L expression of MCM3 protein were mainly expressed in the nucleus, trace expression in the cytoplasm. In order to enhance; group 50mg/L mainly expressed in the cytoplasm, the nucleus has a small amount of expression, the expression intensity is weak. The ELISA results showed that high dose fluoride group 150mg/L, fluoride in 6 months, the expression of MCM3 and the other groups were statistically significant P0.05. in vitro osteoclast culture in 48h fluoride group from 24h to 36h, compared with the control group, osteoclast proliferation activity increased significantly, with statistical significance, P0.05 group; MCM3 antigen in cultured 24h proliferation activity increased, with statistical significance, compared with the control group P0.05; when the interaction of fluoride and MCM3 antigen, 12h ~ 36h in cultured osteoclasts, the most obvious proliferation activity, compared with the control group had statistical significance, P0.05; when the MCM3 antibody effect on osteoclasts cultured in 24h ~ 48h, cell proliferation activity has decreased, compared with fluoride + antigen group, the difference was statistically significant, P0.05. said The MCM3 antigen relative to the antibody has the trend of stimulating the proliferation of osteoclasts at the early stage. With the prolongation of the action time, MCM3 antigen strengthens the proliferation of osteoclasts, and the ability of osteoclast proliferation is also related to MCM3 in the late stage of fluoride exposure.
Conclusion: different doses of fluoride on osteoclasts cultured in vitro expression of MCM3 was different, 2mg/L showed the expression of 10mg/L, promote MCM3 fluoride group, 50mg/L fluoride group significantly inhibited the expression of MCM3, 10mg/L from the fluoride dose began to inhibit the promotion, with gradually weakened along with the increase of the dose, reached in 50mg/L toxicity; high dose fluoride 150mg/L has obvious enhancement effect on expression of MCM3 in vivo, that promotes osteoclast proliferation, its mechanism needs further study; MCM3 in a certain period of time in vitro osteoclast proliferation promoting effect, but the specific mechanism needs further study.

【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R329

【参考文献】