【作者】 张艳丽；

【导师】 巴月；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2018， 硕士

【摘要】 目的通过分析ERα基因启动子区甲基化水平及启动子区多态性与氟暴露地区成人骨组织改变的关系,从表观遗传学和遗传学方面探索其发病机制,为从分子水平探讨氟暴露人群ERα基因与骨代谢的关系提供基本依据。对象与方法1调查对象的选择于2011至2012年,采用横断面研究方法,以河南省开封市饮水型氟中毒地区作为调查点,整群抽取所选调查点18~55岁在当地居住5a以上的人群作为调查对象,共招募到1131人,分别进行问卷调查及体格检查。在横断面研究基础上,根据骨X光片结果,以骨组织异常人群作为病例组,并按年龄(±5岁)、性别约1:1比例进行匹配,抽取骨组织正常人群为对照组,进行病例对照研究,最终纳入研究252人,其中骨组织异常组144人(男51人、女93人),骨组织正常组108人(男50人、女58人),采集生物样本并进行标志物分析。2生物标志的检测数字X线检查仪检测人群骨组织改变情况,检查结果由专业影像医师判定。采用氟离子选择电极法测定尿氟含量;原子吸收分光光度法测定血清钙、镁含量;化学发光免疫分析法测定雌激素含量;甲基化特异荧光定量PCR法测定ERα基因启动子区甲基化水平;TaqMan探针Real-time PCR法检测ERαrs2941740基因型。3数据处理采用Epidata 3.0软件进行数据录入;SPSS 21.0软件进行数据分析。主要统计分析方法有:χ~2检验、独立样本t检验、秩和检验、多重线性回归、二元logistic回归。检验水准α=0.05。结果1骨组织异常组与正常组人群生物标志分析(1)在骨组织异常人群中,有67.9%(72/106)尿氟水平超过1.6 mg/L,而骨组织正常人群中有32.7%(34/106),其差异有统计学意义(P<0.05)。骨组织异常组男性、女性人群尿氟水平分别为(2.01±1.48,2.09±1.59)mg/L,均高于骨组织正常组(1.41±0.86,1.41±1.22)mg/L,差异有统计学意义(P<0.05)。(2)女性人群中骨组织异常组血清镁水平(0.93±0.18)mmol/L高于骨组织正常组(0.85±0.30)mmol/L,差异有统计学意义(P<0.05)。2 ERαrs2941740基因多态性与氟暴露成人骨组织改变的关系(1)骨组织异常组和正常组人群ERαrs2941740基因型分布相似,差异无统计学意义(P>0.05)。(2)校正年龄、性别及尿氟后,以携带CC基因型为参照,携带CT基因型(OR=1.059,95%CI:0.545~2.056)和TT基因型(OR=3.144,95%CI:0.577~17.140)未增加人群骨组织改变的危险性。(3)校正年龄、性别及尿氟后,以携带C等位基因为参照,携带T等位基因未增加人群骨组织改变的危险性(OR=1.333,95%CI:0.770~2.307)。(4)校正年龄和性别后,氟暴露水平是骨组织改变的影响因素(β=0.470,P<0.05);氟暴露与ERαrs2941740基因多态性对人群骨组织改变的影响不存在交互作用(P>0.05)。3 ERαrs2941740基因多态性与氟暴露成人血清标志物的关系(1)在不同氟暴露水平下,携带CC基因型人群血清钙和血清镁水平差异有统计学意义(P<0.05),高氟组携带CC基因型个体血清钙和血清镁水平(2.97±0.86,0.97±0.17)mmol/L均高于低氟组(2.52±1.05,0.88±0.25)mmol/L;而携带CT和TT基因型人群血清钙、血清镁及雌激素差异均无统计学意义(P>0.05)。(2)在相同氟暴露水平下,携带不同基因型人群的血清钙、血清镁及雌激素水平差异均无统计学意义(P>0.05)。4 ERα基因启动子区甲基化水平与氟暴露成人骨组织改变的关系(1)男性人群中,骨组织异常组ERα基因启动子区甲基化水平(4.58±1.53)%低于骨组织正常组(11.75±7.55)%,差异有统计学意义(P<0.05)。(2)校正年龄和性别后,血清钙(β=-0.634)、氟暴露(β=0.552)及甲基化水平(β=-4.986)对骨组织改变有显著影响(分别为P<0.05)。(3)男性人群中,甲基化与氟暴露水平成负相关(r=-0.221,P=0.027),甲基化与血清钙、血清镁及雌激素水平无显著相关(P>0.05);女性人群中,甲基化与血清钙、血清镁、雌激素及氟暴露水平均无显著相关(P>0.05)。(4)相同氟暴露水平下携带不同ERαrs2941740基因型人群启动子区甲基化水平比较差异均无统计学意义(P>0.05);不同氟暴露水平携带相同ERαrs2941740基因型人群启动子区甲基化水平比较差异均无统计学意义(P>0.05)。(5)校正年龄和性别后,氟暴露水平与ERαrs2941740基因多态性对ERα基因启动子区甲基化水平的影响不存在交互作用(P>0.05)。结论1 ERα基因启动子区甲基化水平可能是环境氟暴露对男性人群骨组织影响的重要环节。2血清钙及ERα基因启动子区甲基化水平升高是氟暴露地区人群骨组织改变的保护因素。3未观察到ERαrs2941740基因多态性与氟暴露的交互作用对ERα基因启动子区甲基化水平及成人骨组织改变的影响。更多还原

【Abstract】 Objective This study analyzed the involvement of the methylation level and rs2941740 polymorphism in the promoter region of the ERα gene with bone changes in adults exposed to fluoride from the perspective of epigenetics and genetics,and provided a basis for exploring the relationship among fluoride exposure,ERα gene and bone metabolism at the molecular level.Materials and methods1 Study population The cross sectional study was conducted in endemic fluorosis villages in Kaifeng City of Henan Province from 2011 to 2012.A total of 1131 local residents were recruited via cluster sampling,who were aged from 18 to 55 years old and lived in the villages for more than 5 years.Then questionnaire collection and physical examination were conducted.Based on the cross sectional study,residents with bone tissue abnormalities in X-ray radiographs were selected as the case group.Matched by age(±5 years old)and gender,residents with bone tissue normalities in X-ray radiographs were taken as the control group according to the proportion of about 1:1,and then a case-control study was conducted.A total of 252 subjects were recruited including 144 subjects in bone change cases and 108 subjects in controls.Biological samples including fasting blood and morning urine were collected and biomarkers were analyzed.2 Detection of biomarker X-ray diagnosis was performed to detect bone changes by a medical professional in a certified hospital.Urinary fluoride content was detected by fluoride ion selective electrode method.The levels of serum calcium and magnesium were determined by atomic absorption spectrophotometry.Chemiluminescence immuno-assay was used to detect the level of serum estradiol.The level of methylation in promoter region of ERα gene was detected by quantitative methylation specific polymerase chain reaction.Genotypes were identified by Taqman probe and Real-time PCR.3 Statistical analysis The data were entered into the database using Epidata 3.0 software.Statistical analysis was performed using the SPSS statistical software package,version 21.0.The main statistical methods were Chi-square test,t test for independent samples,rank sum test,multiple linear regression and binary logistic regression.It was considered statistically significant when P≤ 0.05.Results1 Analysis of biomarkers in bone change cases and controls(1)In bone change cases,there were 67.9%(72/106)subjects with urinary fluoride level exceeding 1.6 mg/L,and 32.7%(34/106)subjects in controls.The urinary fluoride level in cases was higher than that in the controls,and the difference was statistically significant(P<0.05).The urinary fluoride level in cases was higher than that in the controls,and the difference was statistically significant(P<0.05).(2)In the female subjects,the serum magnesium level in cases was higher than that in controls,and the difference was statistically significant(P<0.05).2 Relationship between ERα gene rs2941740 polymorphism and bone changes in adults exposed to fluoride(1)The distribution of ERα rs2941740 genotype was similar between the bone change cases and controls,and the difference was not statistically significant(P>0.05).(2)After adjusted for age,gender and urinary fluoride,compared to the CC genotype,subjects carrying CT genotype(OR=1.059,95% CI: 0.545-2.056)and TT genotype(OR=3.144,95% CI: 0.577-17.140)did not increase the risk of bone changes.(3)After adjusted for age,gender and urinary fluoride,carrying the C allele as reference and subjects carrying the T allele did not increase the risk of bone changes(OR=1.333,95% CI: 0.770-2.307).(4)After adjusted for age and gender,the level of fluoride exposure was the influencing factor of bone changes(β=0.470,P<0.05).There was no interaction between fluoride exposure and ERα gene rs2941740 polymorphism on bone changes(P>0.05).(1)There were significant differences in serum calcium and serum magnesium levels among subjects with CC genotype in the different fluoride exposure groups(P<0.05).Serum calcium and serum magnesium levels were higher in the high fluoride exposure group than that in the low fluoride exposure group.However,no significant differences were observed in serum calcium,serum magnesium and estrogen levels in subjects with CT and TT genotypes in different fluoride exposure groups(P>0.05).(2)The serum calcium,serum magnesium and estrogen levels were no significant differences in the same fluoride exposure group when subjects carrying different genotypes of ERα gene rs2941740(P>0.05).4 Relationship between the ERα gene methylation level in promoter region and bone changes in adults exposed to fluoride(1)In the male subjects,the methylation level in the promoter region of ERα gene in bone change cases was lower than that in controls,and the difference was statistically significant(P<0.05).(2)After adjusted for age and gender,the levels of serum calcium(β=-0.634),fluoride exposure(β=0.552)and methylation(β=-4.986)had significant effects on bone changes(P<0.05,respectively).(3)The methylation level in male subjects was negatively correlated with the urinary fluoride(r=-0.221,P=0.027).There were no significant correlation between methyla-tion and the levels of serum calcium,serum magnesium,and estrogen in both male and female subjects(P>0.05,respectively).(4)For subjects who carrying the different genotypes of ERα gene rs2941740 locus,there was no significant difference of the promoter methylation level in the same fluoride exposure group(P>0.05).No significant difference was found in the ERαgene promoter methylation level between the high fluoride exposure group and low fluoride exposure group who carrying the same genotype of ERα rs2941740(P>0.05).(5)After adjusted for age and gender,no interaction was observed between fluoride exposure level and ERα rs2941740 genotypes on the methylation level in the ERαgene promoter region(P>0.05).3 Relationship between ERα rs2941740 polymorphism and serum biomar-kers in adults exposed to fluorideConclusion 1 In the male subjects,the ERα gene methylation level in promoter region may be an important link in the impact of environmental fluoride exposure on bone changes.2 Increased levels of serum calcium and ERα gene methylation in promoter region were protective factors for bone changes in adults exposed to fluoride.3 No interaction was found between ERα gene rs2941740 polymorphism and fluoride exposure on the methylation level in ERα gene promoter region and bone changes.更多还原