孕期p,p’-DDE暴露对仔鼠胰岛细胞IGF2/H19基因印记和胰岛功能的影响及叶酸的干预作用

Effects of prenatal exposure to p, p’-DDE on IGF2/H19 gene imprinting and islet function in rat offspring and the intervention effect of folic acid

[背景]糖尿病是在全球都具有高发病率的一种慢性疾病,而我国确诊的糖尿病人数已经超过1亿。多项研究表明,2,2-双(对氯苯基)——1,1-二氯乙烯(p,p'-DDE)暴露可以干扰葡萄糖代谢以及胰岛素分泌过程,与Ⅱ型糖尿病发病率升高之间具有相关性。另外,孕期p,p'-DDE暴露可以引起子代糖耐量受损,致子代糖尿病敏感性升高。[目的]研究p,p'-DDE孕期暴露对子代SD大鼠胰岛细胞胰岛素样生长因子-2(IGF2)/H19基因印记和胰岛功能的影响,以及甲基供体叶酸在此过程中的干预作用。[方法]采用逆行灌注法提取原代胰岛细胞并进行纯度、存活率与胰岛素分泌功能鉴定。将8~10周性成熟SD大鼠合笼交配,孕鼠随机分为对照组、p,p'-DDE染毒组和叶酸干预组,每组8只。p,p'-DDE染毒组和叶酸干预组于孕第8~15天每天按5 mL/kg(以体重计)连续灌胃给予20 mg/mL p,p'-DDE;对照组给予等容积的玉米油;叶酸干预组整个孕期进食添加3.5 mg/kg叶酸的饲料。孕鼠自由分娩,每窝保留雌雄仔鼠各4只。各组仔鼠21 d断乳后均摄食普通饲料。称取出生后1周、3周及8周仔鼠体重。提取出生后8周仔鼠胰岛细胞DNA和RNA,分别用亚硫酸氢盐法和实时荧光定量PCR法检测胰岛细胞IGF2/H19基因印记调控区域甲基化水平以及IGF2和H19 mRNA表达水平。出生后8周的仔鼠进行葡萄糖耐量试验,测定空腹及灌胃后15、30、60、120 min尾静脉血中的血糖水平。同时,灌胃前及灌胃后2 h进行眼眶采血,ELISA测定血清中的胰岛素水平。[结果]提取的原代胰岛细胞经鉴定,纯度为(90±5)%,存活率大于90%,具有胰岛素分泌功能。p,p'-DDE染毒组仔鼠IGF2/H19基因印记调控区甲基化水平[(51.5±3.8)%]低于对照组[(55.9±3.3)%](P=0.031),与叶酸干预组[(52.8±1.9)%]相比差异无统计学意义。p,p'-DDE染毒组仔鼠IGF2 mRNA相对表达水平[(265.4±70.6)%]高于对照组(100%)(P=0.002)和叶酸干预组[(101.8±65.9)%](P=0.002);各组H19 mRNA相对表达水平差异无统计学意义。3组仔鼠在出生后1周、3周与8周体重差异均无统计学意义。p,p'-DDE染毒组仔鼠灌胃后15 min血糖水平[(10.89±1.17)mmol/L]高于对照组[(9.29±1.18)mmol/L](P=0.026)和叶酸干预组[(9.25±0.95)mmol/L](P=0.022)。3组仔鼠空腹及餐后2 h血清胰岛素水平差异均无统计学意义。[结论] p,p'-DDE孕期暴露可降低子代SD大鼠胰岛细胞IGF2/H19的印记调控区甲基化水平,上调IGF2 mRNA转录水平,诱导子代胰岛细胞功能受损。叶酸在该过程中具有一定的干预作用。

[Background] Diabetes mellitus is a chronic disease with high incidence all over the world and the number of diagnosed diabetes in China has exceeded 100 million. Several studies have shown that p, p'-dichlorodiphenyldichloroethylene (p, p'-DDE) exposure can interfere with glucose metabolism and insulin secretion, and has a correlation with the increased incidence of type 2 diabetes mellitus. In addition, exposure to p, p'-DDE during pregnancy can cause impaired glucose tolerance and induce diabetes in offspring.[Objective] This study aims to investigate the effects of prenatal exposure to p, p'-DDE on insulin like growth factor 2 (IGF2)/H19 gene imprinting and islet function in the offspring of SD rats, and to study whether folic acid as a methyl donor has an intervention effect on the process.[Methods] The primary islet cells were isolated by retrograde perfusion method, and were tested for purity, survival rate, and insulin secretion function. Sexually mature SD rats at 8-10 weeks old were caged and mated, and pregnant rats were randomly divided into three groups:control group, p, p'-DDE group, and folic acid group, with eight rats in each group. On gestational day 8-15, the rats in the p, p'-DDE group and the folic acid group were given p, p'-DDE (20 mg/mL) at 5 mL/kg (body weight) by gavage;the rats in the control group were given the same volume of corn oil;the rats in the folic acid group were fed a diet supplemented with folic acid (3.5 mg/kg). At birth, pups were culled to four males and four females in each litter and were fed an ordinary diet after weaning. Weights of the pups were weighed in the first, third, and eighth weeks after birth. Eight weeks after birth, DNA and RNA were extracted to detect the methylation level of IGF2/H19 imprinting region by bisulfite genomic sequencing and the expression levels of IGF2 and H19 mRNA by real-time quantitative PCR. The pups at 8 weeks old were subject to glucose tolerance test. The blood glucose levels in the caudal vein were measured before and 15, 30, 60, and 120 min after intragastric administration. At the same time, the serum insulin levels in the blood of orbit were determined by ELISA before and 2 h after intragastric administration.[Results] The purity was (90±5)%, the survival rate was more than 90%, and the primary islet cells secreted insulin. The methylation level of IGF2/H19 imprinting region in the p, p'-DDE group offspring[(51.5±3.8)%] was lower than that in the control group offspring[(55.9±3.3)%](P=0.031), but had no significant difference compared with the folic acid group offspring[(52.8±1.9)%]. The expression level of IGF2 mRNA in the p, p'-DDE group offspring[(265.4±70.6)%] was higher than those in the control group offspring (100%)(P=0.002) and the folic acid group offspring[(101.8±65.9)%](P=0.002). No significant difference was found in H19 mRNA expression level among the three groups. There was also no significant difference in body weight in the first, third, and eighth weeks among the three groups. The blood glucose level at 15 min after intragastric administration in the p, p'-DDE group offspring[(10.89±1.17) mmol/L] was higher than those in the control group offspring[(9.29±1.18) mmol/L](P=0.026) and the folic acid group offspring[(9.25±0.95) mmol/L](P=0.022). There was no significant difference in serum insulin level before and 2 h after intragastric administration among the three groups.[Conclusion] Prenatal exposure to p, p'-DDE could reduce the methylation level of IGF2/H19 imprinting region and up-regulate the transcription of IGF2 mRNA, and then impair islet function in the offspring of SD rats. Folic acid plays an intervention role in this process.