【作者】 宋玉娥；

【导师】 刘克俭；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2010， 硕士

【摘要】 氟性骨损伤(fluoride bone injury)是由于机体长期摄入过量氟而引起的以骨代谢过程紊乱为主要病理特征的慢性代谢性骨病。氟性骨损伤骨周化骨是一种特殊的异位骨化形式(heterotopic ossification),其临床表现主要为广泛性骨质增生硬化、肌腱韧带钙化等,是氟性骨损伤患者致残、致瘫的重要原因。通过大量的体外细胞培养观察和动物实验,有充分依据证明成骨细胞活跃和骨转化加速是氟性骨损伤的特征性病变。骨代谢局部网络相关因子的调控作用是氟性骨损伤骨转换加速的机制之一。锌指结构转录因子Osterix参与成骨细胞的分化和骨形成,特异表达于成骨细胞分化和钙化过程中,促进成骨细胞的分化、成熟、膜成骨、软骨化骨及矿化过程,调控许多重要成骨基因的表达,如骨钙素(osteocalcin)、骨粘素(osseomucin)、骨桥蛋白(osteopontin)、骨唾液酸蛋白(bone sialoprotein)和Ⅰ型胶原蛋白(collagen typeⅠ)等。研究证明Osterix具有促进成骨细胞分化和异位骨化的作用。核心结合因子α1(core binding factor alpha 1,Cbfa1,又称Runx2,)是成骨细胞开始分化的最早最特异的标志性基因,高限制性地表达于骨中。因此,Cbfa1和Osterix是成骨过程中必不可缺的转录因子。绝大数疾病的发生都是环境和基因相互作用的结果。本研究通过血清Osterix、Cbfa1蛋白浓度的检测,分析氟暴露人群及氟性骨损伤骨周化骨中Osterix、Cbfa1蛋白浓度的改变,寻找氟性骨损伤骨周化骨的早期损伤效应的监测指标;探讨Osterix基因与氟性骨损伤骨周化骨的易感性,阐述Osterix基因在氟性骨损伤骨周化骨发生发展中的可能作用,以期为氟性骨损伤的防治提供新的靶点,为氟危害的进一步研究提供理论依据。目的:通过血清Osterix和Cbfa1蛋白浓度以及Osterix基因外显子2(exon2 141-1042 bp)的突变检测,分析氟暴露及氟性骨损伤骨周化骨中Osterix和Cbfa1蛋白浓度的变化及血氟、尿氟、Osterix、Cbfa1蛋白浓度之间的相关性,构建氟性骨损伤骨周化骨相关危险因素的Logistic回归模型,探讨Osterix基因与氟性骨损伤骨周化骨的易感性以及Osterix在氟性骨损伤骨周化骨发生发展中的可能作用。方法:选取湖北某铝业集团连续工作5年以上男性作业工人,根据所在车间的不同随机选择研究对象,其中包括:电解车间工人176人;铝杆及铝板带车间工人28人;机关及质检工人22人。并在研究对象知情同意的原则下采集生物材料。根据氟斑牙诊断标准及骨骼X线拍片(骨盆正位片及左侧桡骨正位片)判定结果将研究对象划分为:①有氟性骨损伤骨周化骨工人75人(简称骨损伤组),均来自电解车间。并按骨损伤程度将研究对象分为两组:骨损伤轻度组(30人)和骨损伤重度组(45人);②有氟斑牙但无骨周化骨工人22人(简称氟斑牙组),其中电解车间16人,铝杆及铝板带车间4人,机关及质检工人2人;③氟暴露但无氟斑牙且无氟性骨损伤骨周化骨组工人129人,其中,电解车间85人(简称高氟组),铝杆及铝板带车间工人24人(简称低氟组);机关及质检工人20人(简称对照组)。采用ELISA法测定血清中Osterix和Cbfa1蛋白浓度,Logistic回归方法构建氟性骨损伤骨周化骨危险因素的模型;采用HRM、DNA测序方法检测Osterix基因(位于exon2 141-1042bp)的3个SNP位点。结果:1)低氟组Osterix蛋白浓度为(1159.20±387.58)ng/ml,高氟组Osterix蛋白浓度为(743.81±492.41)ng/ml,对照组Osterix蛋白浓度为(860.09±410.98)ng/ml,低氟组Osterix蛋白浓度高于对照组和高氟组,差异有统计学意义(P<0.05);低氟组Cbfa1蛋白浓度为(916.40±360.21)ng/ml,高氟组Cbfa1蛋白浓度为(629.43±430.47)ng/ml,低氟组Cbfa1蛋白浓度高于高氟组,差异有统计学意义(P<0.05);2)氟斑牙组Osterix蛋白浓度为(1216.20±368.89)ng/ml,骨损伤组Osterix蛋白浓度为(802.61±516.55)ng/ml,对照组Osterix蛋白浓度为(860.09±410.98)ng/ml,氟斑牙组Osterix蛋白浓度高于对照组和骨损伤组,差异有统计学意义(P<0.05);氟斑牙组Cbfa1蛋白浓度为(1087.00±287.53)ng/ml,骨损伤组Cbfa1蛋白浓度为(753.28±486.40)ng/ml,氟斑牙组Cbfa1蛋白浓度高于骨损伤组,差异有统计学意义(P<0.05);骨损伤轻度组及重度组Osterix蛋白浓度为(797.88±486.32)ng/ml和(806.72±516.44)ng/ml,低于氟斑牙组Osterix蛋白浓度,差异有统计学意义(P<0.05);骨损伤轻度组及重度组Cbfa1蛋白浓度为(726.38±494.43)ng/ml和(776.68±491.79)ng/ml,低于氟斑牙组Cbfa1蛋白浓度,差异有统计学意义(P<0.05);3)骨损伤组血氟与Osterix和Cbfa1蛋白浓度的相关系数r值分别-0.434、-0.469,尿氟与Cbfa1蛋白浓度的相关系数r值为-0.436,差异有统计学意义(P<0.05);对照组血氟、尿氟与Osterix、Cbfa1蛋白浓度的相关系数r值分别为-0.227、-0.274、-0.075、-0.058,差异无统计学意义(P>0.05);Osterix与Cbfa1蛋白浓度的相关系数r值在骨损伤组及对照组中分别为0.897、0.878,差异有统计学意义(P<0.01);4)在Logistic回归模型中,低浓度的血氟(血氟≤0.17 mg/L及0.17 mg/L<血氟≤0.20 mg/L)与高浓度的血氟(血氟>0.20mg/L)与相比,OR值分别为0.244和0.264(P=0.039、0.033),B值为-1.409和-1.333;低浓度的尿氟(尿氟≤2.00mg/L及2.00<尿氟≤4.00 mg/L)与高浓度的尿氟(尿氟>4.00 mg/L)与相比,OR值分别为0.051和0.086(P =0.000、0.000),B值为-2.978和-2.451;低水平的Osterix蛋白浓度(400.00 ng/ml <Osterix蛋白浓度≤800.00 ng/ml和800.00ng/ml<Osterix蛋白浓度≤1200.00 ng/ml)与高水平的Osterix蛋白浓度(Osterix蛋白浓度>1200.00 ng/ml)相比,OR值分别为18.148、5.746(P =0.044、0.030),B值为2.899和1.749 ;低水平的Cbfa1蛋白浓度(800.00ng/ml<Cbfa1蛋白浓度≤1200.00 ng/ml)与高水平的Cbfa1蛋白浓度(Cbfa1蛋白浓度>1200.00 ng/ml)相比,OR值为0.151(P =0.016),B值-1.892。5)Osterix基因(位于exon2 141-1042bp)三个位点的基因型在损伤对照之间的基因型相同,分布无差异,均为纯合子,分别为CC、GG、CC。结论:1)在低氟暴露、低氟负荷时,Osterix和Cbfa1蛋白浓度升高,而在高氟暴露、高负荷时Osterix和Cbfa1蛋白浓度降低,即随着氟暴露浓度及氟负荷的增加,Osterix、Cbfal蛋白浓度呈降低的趋势;2)机体发生氟斑牙但无骨损伤时,Osterix和Cbfa1蛋白浓度升高,而在骨损伤后Osterix和Cbfa1蛋白浓度降低,即随着氟性骨损伤骨周化骨的发生及损伤程度的加重,Osterix、Cbfal蛋白浓度也呈降低的趋势,Osterix和Cbfa1蛋白浓度降低的拐点可能是氟性骨损伤骨周化骨发生的起点; 3)骨损伤组Osterix、Cbfa1蛋白浓度与血氟、尿氟呈负相关,即Osterix、Cbfa1蛋白浓度随血氟、尿氟的升高呈降低的趋势;而骨损伤组与对照组中Osterix蛋白浓度与Cbfa1蛋白浓度呈正相关,即Osterix蛋白浓度随Cbfa1蛋白浓度升高而升高;4)回归分析中,高血氟水平(血氟>0.20mg/L)和高尿氟水平(尿氟>4.00 mg/L)、高水平的Cbfa1蛋白浓度(Cbfa1蛋白浓度>1200.00 ng/ml)以及Osterix蛋白浓度在大于400.00ng/ml且小于等于1200.00ng/ml的范围时,发生氟性骨损伤骨周化骨的风险增高。5)氟性骨损伤骨周化骨患者中,Osterix基因外显子2上发现碱基的突变及单核苷酸多态性,故锌指结构转录因子Osterix与氟性骨损伤骨周化骨的易感性有待进一步研究。综上所述,在低氟暴露、低氟负荷及机体发生氟斑牙时,Osterix和Cbfa1蛋白浓度升高,而在高氟暴露、高负荷及骨损伤后时,Osterix和Cbfa1蛋白浓度降低,Osterix和Cbfa1蛋白浓度降低的拐点可能是氟性骨损伤骨周化骨发生的起点。因此,我们认为Cbfa1和Osterix的蛋白浓度可以作为氟性骨损伤骨周化骨早期损伤效应的监测指标。更多还原

【Abstract】 Fluoride bone injury is chronic metabolic bone disease caused by long-term excess fluoride intake, which mainly pathological feature is bone metabolism disorder. Heterotopic ossification of fluoride bone injury is a special kind of ectopic osteogenesis, and its mainly clinical manifestation include extensive hyperostosis osteosclerosis, osteomalacia, osteoporosis, and calcification of interosseous membrane, muscle tendon and ligament, and so on, which may be the important factor leading to paralysis and mutilation. The active of osteoblasts and the acceleration of bone turnover are the characteristic lesions of fluoride bone injury according to a wealth of cell culture in vitro and animal experiments. Regulatory effect of the related factors in local bone metabolism network is one of the mechanisms of bone turnover acceleration resulted from excess fluoride.Osterix, a zinc-finger transcription factor, which is connected with osteoblasts and bone formation. It is specifically expressed in the process of osteoblasts differentiation and calcification, and can regulate many important genes expression, such as osteocalcin, bone sialoprotein, collagen type I and so on. Studies found that Osterix could promote the process of osteoblasts differentiation and heterotopic ossification. Core-binding factor-1 (Cbfa1, also known as Runx2) which high-restrictive expression in the bone, is an early and specific marker gene at the beginning of osteoblasts differentiation. Therefore, Osterix and Cbfa1 are the essential transcription factors in the presses of bone formation.Almostly diseases are the interaction between genes and environment. This subject analysis the protein changes of Osterix and Cbfa1 in fluoride exposures, search for the early indexes of bone damage by detecting the serum protein concentration of osterix and Cbfa1; our study also investigate the gene susceptibility of osterix in fluoride bone injury, expound the role of osterix in the process of fluoride bone injury, hoping to provide a new target for the prevention of fluoride bone injury, and providing theory basis for further research.Objective To analysis the changes of Osterix(zinc finger structures transcriptional factor) and Cbfa1 protein levels in the fluoride exposures and heterotopic ossification of fluoride bone injuries, and the correlation between serum fluoride, urinary fluoride and Osterix,Cbfa1 protein levels, to construct Logistic regression model with the associated risk factors of heterotopic ossification of fluoride bone injury, then to study on the susceptibility between heterotopic ossification of fluoride bone injury and Osterix gene, and the role of Osterix in the process of heterotopic ossification in fluoride bone injury by examining the concentrations of Osterix and Cbfa1 protein in serum and the base mutation of 1kb-length-Osterix gene.Methods Male people were chosen who have worked more than five years in one the Aluminum plant in Hubei Province, according to the different work places. Research objects included: 176 workers were chosen from the Potroom, 28 workers were chosen from the Aluminum Rod and Aluminum Strip Departments and 22 workers were chosen from the Organ and Quality Test Departments. Biomaterials were collected under the principle of informed consents. According to the Fluorotic teeth diagnostic criteria and bone X-ray examinationl (pelvic and left radius anterior posterior X-ray examination),the research object will be divided into:①75 workers who had heterotopic ossification of fluoride bone injuries(bone injury group for short), which were from the Potroom; According to the degree of heterotopic ossification in fluoride bone injury, people were divided into two groups: mild-bone injury group (30 workers) and severe-bone injury group (45 workers);②22 workers who just had dental fluorosis but not bone-injury(dental fluorosis group for short),in which 16 worker were from the Potroom, 4 worker were from the Aluminum Rod and Aluminum Strip Departments, and 2 worker were from the Organ and Quality Test Departments;③129 fluoride exposure workers without dental fluorosis or bone-injury, in which 85 worker were from the Potroom(high fluoride group for short), 24 worker were from the Aluminum Rod and Aluminum Strip Departments(low fluoride group for short), and 20 worker were from the Organ and Quality Test Departments(the control group). The concentrations of Cbfa1 and Osterix protein were detected by enzyme-linked immunosorbent assay method. Then used the Logistic regression to construct the model with the associated risk factors of heterotopic ossification of fluoride bone injury. Using HRM and DNA sequencing to detect three SNP sites (located in exon2 141-1042bp)of Osterix gene.Results 1) The concentration of Osterix in the low fluoride group was(1159.20±387.58)ng/ml, but in high fluoride group it was(743.81±492.41)ng/ml, in the control group it was(860.09±410.98)ng/ml, which was higher in the low fluoride group than in the high fluoride group and control group, and the difference had statistical significance(P<0.05);The concentration of Cbfa1 in the low fluoride group was(916.40±360.21)ng/ml, but in high fluoride group it was(629.43±430.47)ng/ml, which was higher in the low fluoride group than in the high fluoride group, and the difference had statistical significance(P<0.05); 2) The concentration of Osterix in dental fluorosis group was(1216.20±368.89)ng/ml, but in bone injury group it was(802.61±516.55)ng/ml, in the control group it was(860.09±410.98)ng/ml, which was higher in the dental fluorosis group than in the bone injury group and control group, and the difference had statistical significance(P<0.05); the concentration of Cbfa1 in dental fluorosis group was(1087.00±287.53)ng/ml, but in bone injury group it was(753.28±486.40)ng/ml, which was higher in the dental fluorosis group than in the bone injury group, and the difference had statistical significance(P<0.05); the protein concentrations of Osterix in mild-bone injury and severe-bone injury group was(797.88±486.32)ng/ml and(806.72±516.44)ng/ml separately, which lower than its concentrations in the dental fluorosis group, and the difference had statistical significance(P<0.05); the protein concentrations of Cbfa1 in mild-bone injury and severe-bone injury group was(726.38±494.43)ng/ml and (776.68±491.79)ng/ml separately, which lower than its concentrations in the dental fluorosis group, and the difference had statistical significance (P<0.05); 3) In the bone-injury group, correlation coefficient (r) between serum fluoride and Osterix, Cbfa1 protein concentration was -0.434 and -0.469 separately(P<0.05), correlation coefficient (r) between urine fluoride and Cbfa1 protein concentration was -0.436(P<0.05). But in the control group, correlation coefficient (r) between serum fluoride, urine fluoride and Osterix, Cbfa1 protein concentration was -0.227, -0.274, -0.075, -0.058 separately, and difference had no statistical significance(P>0.05). Correlation coefficient (r) between Osterix and Cbfa1 protein concentration in bone-injury and control group was 0.897and 0.878 separately(P<0.05); 4) Logistic regression model showed that: compared with the high concentration of serum fluorine(serum fluorine>0.20mg/L), the low concentration of serum fluorine(serum fluorine≤0.17 mg/L and 0.17<serum fluorine≤0.20 mg/L) had statistical significance, OR was 0.244 and 0.264 separately(P=0.039,0.033), B was -1.409 and -1.333 separately; compared with the high concentration of urine fluorine (urine fluorine>4.00mg/L), the low concentration of urine fluorine(urine fluorine≤2.00 mg/L and 2.00<urine fluorine≤4.00mg/L) had statistical significance, OR was 0.051 and 0.086 separately(P=0.000, 0.000), B was -2.978 and-2.451 separately; compared with the high concentration of Cbfa1(Cbfa1>1200.00ng/ml), the low concentration of Cbfa1(800.00ng/ml<Cbfa1≤1200.00ng/ml) had statistical significance, OR was 0.151 (P=0.016), B was -1.892; compared with the high concentration of Osterix (Osterix >1200.00ng/ml), the low concentration of Osterix(400.00ng/ml<Osterix≤800.00ng/ml and 800.00ng/ml<Osterix≤1200.00ng/ml) had statistical significance, OR was 18.148 and 5.746 separately(P=0.044, 0.030), B was 2.899 and1.749 separately; 5) The gene types of the three SNP sites of Osterix gene were CC、GG、CC, and the genotypes were same between the bone-injury and control groups, which were homozygotes, so there was no difference between two groups(P>0.05).Conclusion 1) Osterix and Cbfa1 protein concentration increased with low fluoride exposure and fluoride-loaded, but decreased with high fluoride exposure and high fluoride-loaded, that is to say, Osterix and Cbfa1 protein concentration decreased with the increasing of fluoride exposure and fluoride-loaded; 2) Osterix and Cbfa1 protein concentration increased when the body had dental fluorosis, however, it decreased after bone-injury. In other words, Osterix and Cbfa1 protein concentration decreased with the happening and worse of heterotopic ossification in fluoride bone injuries, so the decreasing turning point of Osterix protein concentration might be the time for the starting of heterotopic ossification in fluoride bone injuries; 3) In the bone-injury group, the correlation between Osterix, Cbfa1 protein concentration and serum fluorine, urine fluorine was negative, so Osterix and Cbfa1 protein concentration reduced with the increase of serum fluorine and urine fluorine; the correlation between Osterix and Cbfa1 was positive, so Osterix protein concentration increased with the rising of Cbfa1; 4) High-level serum fluorine(serum fluorine>0.20mg/L), urine fluorine(urine fluorine>4.00mg/L), Cbfa1 protein concentration(Cbfa1>1200ng/ml), and when Osterix protein concentration was between 400ng/ml and 1200ng/ml, the happening risk of heterotopic ossification of fluoride bone injuries was rising; 5) The SNP sites and gene mutation of Osterix(located in exon2,141-1042bp) were not found in the patients of the heterotopic ossification in fluorine bone injury. So the susceptibility between susceptibility and heterotopic ossification of fluoride bone injury still need further study. To sum up, Osterix and Cbfa1 protein concentration increased with low fluoride exposure, fluoride-loaded and when the body occurs dental fluorosis, but decreased with high fluoride exposure, high fluoride-loaded and after bone-injury, so the decreasing turning point of Osterix protein concentration might be the time for the starting of heterotopic ossification in fluoride bone injuries. So we think Osterix protein concentration can be used as monitoring index of early bone damage in heterotopic ossification of fluoride bone injuries.更多还原