双酚A暴露诱导雌性大鼠性早熟的下丘脑KISS-1和GPR54基因甲基化分子机制探讨  

Molecular mechanism of hypothalamic KISS-1 and GPR54 gene methylation induced by bisphenol A exposure in female rats with precocious puberty

作　　者：罗小娟[1] 曹科[1] 陶明俊[1] 陈小文[2] 张琴[3] 彭刚[4] 陈运生[1] LUO Xiaojuan;CAO Ke;TAO Mingjun;CHEN Xiaowen;ZHANG Qin;PENG Gang;CHEN Yunsheng(Department of Laboratory,Shenzhen Children′s Hospital,Shenzhen,Guangdong;Department of Children Research Institute,Shenzhen Children′s Hospital,Shenzhen,Guangdong;Department of Endocrinology,Shenzhen Children′s Hospital,Shenzhen,Guangdong;Department of Adolescent Gynecology,Shenzhen Children′s Hospital,Shenzhen,Guangdong 518038,China)

机构地区：[1]广东省深圳市儿童医院检验科,518038 [2]广东省深圳市儿童医院儿研所,518038 [3]广东省深圳市儿童医院内分泌科,518038 [4]广东省深圳市儿童医院青春期妇科,518038

出　　处：《重庆医学》2021年第19期3249-3254,共6页Chongqing medicine

基　　金：广东省深圳市科技研发资金(JCYJ20180228175408411);广东省深圳市卫生和计划生育系统科研项目(SZFZ2018054)。

摘　　要：目的探讨双酚A(BPA)暴露诱导雌性大鼠性早熟的下丘脑KISS-1和GPR54基因甲基化分子机制。方法将SD雌性幼鼠(21 d龄)分为4组,即BPA干预组(0.25 mg/kg)、BPA空白对照组(与BPA组同时处死)、17β-雌二醇组(E2,0.1 mg/kg)、溶剂对照组(直至大鼠出现阴道开口再处死),每天以灌胃方式持续给药10 d,以大鼠阴道开口时间(VOD)为观察终点(VOD当天处死),BPA干预组阴道开口当天处死时,按1∶1比例处死BPA空白对照组。收集大鼠卵巢和下丘脑组织,采用real-time PCR方法检测卵巢ERα、ERβmRNA和下丘脑KISS-1、GPR54 mRNA相对表达水平,焦磷酸测序PCR方法检测KISS-1、GPR54基因甲基化水平。结果BPA干预组和E2组VOD较溶剂对照组提前(P<0.05),BPA空白对照组处死时未出现阴道开口,BPA干预组下丘脑KISS-1、GPR54 mRNA表达水平明显高于BPA空白对照组(P<0.05),但与溶剂对照组比较,差异无统计学意义(P>0.05)。E2组下丘脑KISS-1 mRNA和卵巢ERβmRNA表达水平明显低于溶剂对照组(P<0.05)。BPA干预组、BPA空白对照组、E2组和溶剂对照组4组间比较,下丘脑KISS-1基因CpG位点甲基化水平差异无统计学意义,而GPR54基因CpG位点甲基化水平存在显著性差异,其中BPA干预组、E2组和溶剂对照组GPR54片段1 CpG位点甲基化水平显著低于BPA空白对照组(P<0.05),且其甲基化水平与VOD呈正相关(rs=0.245,P=0.009)。BPA干预组GPR54片段2 CpG位点甲基化水平显著低于溶剂对照组,而E2组卵巢ERβ基因甲基化水平显著高于溶剂对照组(P<0.05)。结论BPA暴露可导致雌性幼鼠阴道开口(性早熟)提前,其下丘脑GPR54基因甲基化水平改变可能是毒性机制之一。Objective To investigate the molecular mechanism of hypothalamic KISS-1 and GPR54 gene methylation in female rats induced by bisphenol A(BPA)exposure.Methods The female Sprague-Dawley(SD)rats(21-days-old)were randomly divided into four groups:the BPA intervention group(0.25 mg/kg),the BPA blank control group(executed at the same time as the BPA intervention group),the 17β-estradiol group(E2,0.1 mg/kg)and the solvent control group(executed until vaginal opening appeared).The rats were given the intragastric administration for 10 consecutive days,and the vaginal opening day(VOD)was used as the observation end point.The rats were sacrificed on the day of vaginal opening.According to the 1∶1 proportion,the rats of the blank control group were sacrificed during the death of the BPA group.The ovary and hypothalamus tissues of the rats were collected,the ovarian ERα,ERβand hypothalamic KISS-1 and GPR54 gene mRNA expression were measured by the real-time PCR.The gene methylation was detected by the pyrosequencing PCR.Results The VOD of the BPA intervention group and the E2 group was earlier than that of the solvent control group(P<0.05),and there was no vaginal opening in the BPA blank control group at the time of death.The hypothalamic KISS-1 and GPR54 mRNA expressions in the BPA intervention group were significantly higher than that in the BPA blank control group(P<0.05),but when compared with the solvent control group,there was no statistical significance(P>0.05).The mRNA expression levels of the KISS-1 in the hypothalamus and ERβin the ovary in the E2 group were significantly lower than those in the solvent control group(P<0.05).There was no statistical difference in the methylation level of the CPG locus of the KISS-1 gene in the hypothalamus between the BPA intervention group,the BPA blank control group,the E2 group and the solvent control group,while there was a significant difference in the methylation level of the GPR54 gene.The methylation level of the GPR54 fragment 1 CPG in the BPA intervention group,th

关 键 词：双酚A 阴道开口时间 KISS-1/GPR54基因 甲基化 雌性大鼠 性早熟 

分 类 号：R114[医药卫生—卫生毒理学]