孕鼠交通性污染物暴露对子代雄性睾丸DNA甲基化模式的影响

发布时间：2019-06-26 18:53

【摘要】：目的:本文拟通过建立母鼠孕期暴露于交通性污染物染毒模型,研究孕鼠暴露于交通性污染物后对其子代生殖系统的影响。同时借助简化表观亚硫酸氢盐测序技术从表观遗传学的角度分析交通性污染物对子代雄性生殖系统的影响机制,揭示交通性污染物导致雄性生育能力下降的作用靶点和机理,为寻找男性生殖功能降低的原因和治疗男性不育症提供实验与理论依据。方法:30只孕鼠(C57BL/6J种系)随机分为对照组和暴露组,每组各15只。暴露组孕鼠置于重庆市主城区某一车流量较大的隧道内,从D4-D14每天持续暴露8h。其余时间暴露组与对照组孕鼠均分笼饲养于重庆医科大学动物实验中心普通级,温度(22±1)℃,相对湿度40%-60%,自由进食和饮水。D18后将孕鼠单笼喂养直至其自然分娩。子代雄性小鼠出生后正常饲养至性成熟(55日龄)。在暴露组染毒的D4、D8、D12、D14对隧道交通性污染物NOX、TSP、PM2.5、PM10和噪声进行采集和检测。染毒结束后观察孕鼠流产情况,子代小鼠生殖系统脏器系数,小鼠精子数量、畸形率、小鼠睾丸组织病理学变化。两组各取3只雄性小鼠睾丸进行RRBS甲基化测序并用Real-time PCR、Western Bolt方法检测目的基因Rpe、Aldh7a1的m RNA和蛋白表达水平。结果:隧道交通性污染物NOX(0.079±0.147 mg/m3)、TSP(0.61±0.049 mg/m3)、PM2.5(141±23.45 mg/m3)、PM10(206±20.78 mg/m3)和噪声[77.60±7.05 d B(A)]均超过对照组,差异有统计学意义(P0.05),两组SO2浓度差异没有显著性(P0.05)。暴露组孕鼠流产率(66.67%)高于对照组。暴露组雄鼠精子数量(9.70±5.54′107/g)少于对照组(12.77±2.72′107/g),差异有统计学意义(P0.05),其体质量、睾丸和附睾脏器系数、精子畸形率与对照组相比,无统计学改变(P0.05)。电镜结果显示,暴露组雄鼠睾丸组织中生精小管之间空隙大,管腔内出现较多空泡。与对照组相比,暴露组子代雄鼠睾丸基因组有34个基因上调,81个基因下调。Rpe、Aldh7a1基因分别调控糖酵解途径和丙酮酸代谢过程。与对照组相比暴露组Rpe、Aldh7a1基因m RNA和蛋白相对表达量减少(P0.05)。结论:孕鼠交通性污染物暴露可使子代雄鼠精子数量下降、睾丸组织形态学发生一定程度的改变,也可引起子代雄鼠生殖系细胞表观基因组发生改变。Rpe、Aldh7a1基因下调而导致的氨基酸合成通路受到影响可能是导致子代雄鼠生殖系统损害的原因之一。
[Abstract]:Aim: to establish a model of maternal exposure to traffic pollutants during pregnancy and to study the effects of maternal exposure to traffic pollutants on the reproductive system of their offspring. At the same time, with the help of simplified apparent bisulfite sequencing technique, the influence mechanism of traffic pollutants on male reproductive system of offspring was analyzed from the point of view of epigenetics, and the target and mechanism of male fertility decline caused by traffic pollutants were revealed, which provided experimental and theoretical basis for finding out the causes of male reproductive function decline and treating male sterility. Methods: 30 pregnant rats (C57BL/6J strain) were randomly divided into control group (n = 15) and exposure group (n = 15). The pregnant rats in the exposure group were exposed to D4-D14 for 8 hours per day in a tunnel with large traffic flow in the main urban area of Chongqing. For the rest of the time, the pregnant rats in the exposure group and the control group were fed in cages in the animal experimental center of Chongqing Medical University, the temperature was (22 卤1) 鈩，

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