贵州省燃煤型氟中毒病区人群髓过氧化物酶基因mRNA表达水平及启动子区转录调控研究

A study of mRNA expressi on and transcription regulation in the promoter region of myeloperoxidase gene from a population living in the area with coal-burning endemic fluorosis in Guizhou Province

目的：探讨髓过氧化物酶（Myeloperoxidase， MPO）基因遗传变异与燃煤型氟中毒发病机制的相关性，了解改灶和健康教育等综合干预措施对病区人群的健康影响。方法2007年，选择贵州省毕节市燃煤型地方性氟中毒重病区人群，在没有进行改炉灶的鸭池镇，选择150名氟斑牙患者为氟中毒非干预组；在已进行改灶2年的长春镇，选择150名氟斑牙患者为氟中毒干预组；在非地方性氟中毒病区贵州省长顺县白云山镇，选择150名村民作为对照组。采用实时荧光定量PCR方法，检测各组人群外周血白细胞中MPO mRNA 的表达。体外培养人肝癌细胞株HepG2，构建MPO-463G/A启动子序列报告基因质粒pGL3-A、pGL3-G；pGL3-A、pGL3-G、pGL3-Basic （空白对照）、 pGL3-Control （阳性对照）分别和内参质粒 pRL-TK 共转染 HepG2细胞，即HepG2细胞分为pGL3-A 组、pGL3-G 组、pGL3-Basic 组、pGL3-Control 组。利用双荧光素酶报告基因检测分析MPO基因启动子区-463G/A位点点突变对基因转录活性的影响。结果氟中毒非干预组人群外周血白细胞中MPO mRNA表达（0.054±0.003）高于氟中毒干预组和对照组（0.019±0.003、0.019±0.004，P均＜0.05），而氟中毒干预组MPO mRNA 表达与对照组比较差异无统计学意义（P〉0.05）；MPO基因-463G/A位点发生遗传变异后，pGL3-G 组荧光素酶表达（0.7534±0.0866）高于 pGL3-A 组（0.4900±0.0223，P 〈0.05）。结论 MPO基因启动子区-463G/A位点A等位基因可能是燃煤型氟中毒的保护因素。综合干预措施在防治地方性氟中毒方面有一定作用。

Objective To explore the correlation between myeloperoxidase （MPO） genetic variation and coal-burning endemic fluorosis, and to understand the influence of integrated intervention including stove changes and health education on people’s health in the area. Methods In 2007, coal-burning endemic fluorosis disease areas were selected in Bijie City, Guizhou Province. No stove changes in Yachi Town, 150 patients with dental fluorosis were selected as fluorosis non-intervention group, and the intervention group was 150 patients in Changchun Town where the stoves were changed 2 years ago. The population in control group was selected in an area with non-endemic fluorosis in Changshun County. The mRNA expressions of MPO in leukoxytes were detected by real-time PCR. HepG2 cells were cultured in vitro and divided into four groups： pGL3-A group, pGL3-G group,＆amp;nbsp;pGL3-Control group and pGL3-Basic group. pGL3-A and pGL3-G were recombinant plasmid, while pGL3-Basic as a blank control and pGL3-Control as a positive one. The internal reference plasmid pRL-TK co-transfected the HepG2 cells with pGL3-G, pGL3-A, pGL3-Basic and pGL3-Control, respectively. The influence of sudden change of MPO gene promoter on the gene transfection activity was evaluated by a dual luciferasereporter gene system. Results The expression level of MPO mRNA in peripheral blood leukocytes in non-intervention group（0.054 ± 0 . 003 ） were higher than control and intervention groups （0.019 ± 0.004,0.019 ± 0.003, all P〈0.05）, and no significant change was found between intervention group and control group（P〉0.05）. After the MPO-463G/A locus genetic variation occured, the luciferase reporter gene expression level of the recombinant plasmid pGL3-G（0.753 4 ± 0.086 6） was higher than that of the pGL3-A（0.490 0 ± 0.022 3, P 〈 0.05）. Conclusions The study on MPO gene promoter-463G/A locus has prompted that MPO gene allele may be a protective factor to coal-burning fluorosis. The integrated interventions have a role in the prevention and treatment of endemic fluorosis.