氟化钠对人成骨细胞CyclinD1基因启动子区甲基化及其转录表达的影响  

Effects of sodium fluoride on DNA methylation and expression of CyclinD1 gene in human osteoblasts

作　　者：严威敏 廖玉丹[1] 罗鹏[1] 潘雪莉[1] 

机构地区：[1]贵州医科大学公共卫生学院环境污染与疾病监控教育部省部共建重点实验室,贵州贵阳550025

出　　处：《环境与健康杂志》2016年第4期292-295,共4页Journal of Environment and Health

基　　金：国家自然科学基金(81260418);贵州省优秀科技教育人才省长基金(黔省专合字[2011]54号)

摘　　要：目的构建人原代成骨细胞氟中毒模型,检测不同剂量氟化钠对人成骨细胞中CyclinD1基因启动子区甲基化、mRNA转录及蛋白表达的影响,从细胞周期调控基因的表观遗传学角度探索氟中毒发生机制。方法在知情同意原则下,收集贵州航空工业集团贵阳医院骨科外伤手术健康人(车祸)髂骨或股骨松质骨。采用酶消化法分离人原代成骨细胞,以碱性磷酸酶及钙化结节染色进行细胞鉴定;以0、125、250、500及1 000μmol/L氟化钠处理细胞72 h。以硫化测序PCR法(bisulfite sequencing PCR,BSP)检测CyclinD1基因启动子区甲基化状态;以实时荧光定量PCR(QT-PCR)检测CyclinD1基因转录mRNA相对表达量;以免疫印迹法(Western-blotting)检测CyclinD1蛋白表达。结果分别以0、125、250、500及1 000μmol/L氟化钠处理细胞72 h后,CyclinD1基因启动子区未检测到甲基化;CyclinD1基因mRNA转录相对表达量在0、125、250、500及1 000μmol/L Na F剂量组分别为0.414±0.093、0.742±0.089、0.796±0.122、1.114±0.260、1.140±0.171,NaF剂量组均高于对照组(F=18.89,P<0.05)。CyclinD1蛋白相对表达量分别为0.304±0.014、0.395±0.020、0.511±0.042、0.565±0.028、0.719±0.047,NaF剂量组均高于对照组(F=71.80,P<0.05)。CyclinD1基因转录mRNA及其蛋白表达随氟化钠剂量的升高均相应上升(P<0.05)。结论氟化钠上调人成骨细胞中CyclinD1基因转录与表达,是氟致人成骨细胞增殖能力及细胞周期时相分布发生改变的重要分子机制之一,但未观察到CyclinD1基因启动子区甲基化参与其表达上调过程。Objective To establish an in vitro model of fluorosis with human primary osteoblasts and to detect the influences of different doses of sodium fluoride on methylation,transcription and expression of CyclinD1,then to study fluorosis mechanism from the epigenetic perspective of the cell cycle regulation-related gene.Methods Under the principle of informed consent,ilium and femoral cancellous bones were collected in orthopedic surgery healthy people(car accident) from Guiyang Hospital of Guizhou Aviation Industry Group.Human primary osteoblasts were isolated by enzyme digestion,and identified by morphology,alkaline phosphatase staining and alizarin red staining.The osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L Na F for 72 h,the methylation status of CyclinD1 promoter region was detected by bisulfate-sequencing polymerase chain reaction(BSP).The m RNA transcription and the protein expression of CyclinD1 were detected by realtime quantitative PCR and Western blotting.Results Among the groups of osteoblasts treated with 0,200,400,800 and 1 000 μmol/L for 72 h,no DNA methylation was observed in promoter region of CyclinD1.Average levels of CyclinD1 m RNA were 0.414±0.093,0.742±0.089,0.796±0.122,1.114±0.260,1.140±0.171,and their levels in the fluoride-exposed groups were all significantly higher than the control group(F =18.89,P <0.05).Average levels of CyclinD1 protein were,0.304 ±0.014,0.395 ±0.020,0.511 ±0.042,0.565 ±0.028,0.719±0.047,their levels in the fluoride-exposed groups were all significantly higher than the control group(F=71.80,P <0.05).The levels of CyclinD1 m RNA and protein were both increased as sodium fluoride doses increased(P <0.05).Conclusion Fluoride can upregulate the expression of CyclinD1 m RNA and protein in human osteoblasts,which may be one of the mechanisms of cell proliferation change and cell cycle disorder in skeletal fluorosis,the effect of DNA methylation on this process is not seen in the present study.

关 键 词：氟 人成骨细胞 CYCLIND1基因 DNA甲基化 

分 类 号：R599.1[医药卫生—内科学]