【作者】 韩华；

【导师】 李岩；

【作者基本信息】 遵义医学院 ， 卫生毒理学， 2009， 硕士

【摘要】 目的:探讨利谷隆致雄性仔鼠的生殖毒性及其机制。方法:孕期妊娠第13-18天时用花生油溶解利谷隆(0、50、100、150、200mg/kg)灌胃染毒,于孕期妊娠(GD)20天各组解剖数只孕鼠,提取雄性胎鼠尿生殖结节和睾丸做病理切片;于雄性子代出生后28d测量肛殖距(anogenital distance,AGD)和称重;于雄性子代出生后第2天用酶联免疫吸附试验(enzyme-linked immunosorbentassay,ELISA)检测睾酮激素水平;在胚胎20d解剖孕鼠,提取雄性胎鼠睾丸做电镜观察睾丸间质细胞的超微结构的变化、免疫组织化学检测睾丸间质细胞中P450scc、3β-HSD、PCNA蛋白,AR的表达情况,用实时荧光定量的RT-PCR实验检测睾丸间质细胞中P450c17、17β-HSD、PCNA、AR基因表达情况。结果:1.各组之间雄性仔鼠体重无明显差异。在一定的剂量范围内,随着染毒剂量的增加,用药组AGD随之有缩短的趋势。2.LIN染毒组与空白LIN 0mg/kg对比,病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.LIN染毒组雄性子代出生后第2d睾酮激素水平较LIN 0mg/kg降低。4.电镜观察结果:睾丸间质细胞粗面内质网扩张,线粒体肿胀。5.免疫组织化学结果:在睾丸间质细胞中P450scc酶、3β-HSD、PCNA蛋白表达降低(P均＜0.05),AR蛋白表达与LIN 0mg/kg相近(P＞0.05)。6.实时荧光定量的RT-PCR实验显示:在睾丸间质细胞中P450c17基因,17β-HSD、PCNA基因表达减弱(P均＜0.05),AR基因表达与LIN 0mg/kg相近(P＞0.05)。结论:孕期暴露LIN可透过胎盘屏障,对雄性仔鼠造成如下影响:1.各染毒组与LIN0mg/kg之间体重无差异。在一定的剂量范围内,随着染毒剂量的增加,染毒组AGD随之有缩短的趋势。2.染毒组与空白LIN 0mg/kg对比病理见睾丸索变细,睾丸内各细胞空泡变性,核固缩增多。3.ELISA显示:除染毒组50mg/kg外,其以上剂量可使睾酮激素水平降低,可见睾酮激素分泌水平与染毒剂量之间具有剂量效应关系。4.电镜观察结果证实了LIN能够引起大鼠睾丸间质细胞粗面内质网扩张,线粒体肿胀,从而导致睾酮激素的合成障碍。5.免疫组化显示LIN能够降低睾丸间质细胞中合成睾酮代谢酶P450scc和3β-HSD,PCNA酶蛋白的活性表达,从而影响睾酮的产生。6.实时荧光定量的RT-PCR实验显示:LIN降低了P450c17,17β-HSD、PCNA基因在睾丸间质细胞中在表达,利谷隆对雄性生殖系统的影响可能是通过影响睾酮生成过程中的关键酶造成睾酮生成减少的。更多还原

【Abstract】 Objective:To explore reproductive toxicity and mechanism of linuron on F1 male rats.Method:Pregnant female rats were exposed to LIN(LIN was dissolved to peanut oil) at doses of 0,50,100,150 and 200mg/kg by body weight,respectively,during the period of gestation day(GD) 13 to 18.The fetuses of two rats from each group were anatomized on GD20 to extract genital tubercle(GT) and testis to make pathological section.The datas of AGD and weight were observed on PND28.We detected the level of testosterone with ELISA in PND2 on male offspring.Pregnant SD rats were sacrificed on GD 20.we extracted testis to observe the inner constitution change of cell of Leydig by electron microscope.Moreover,we have done experiment in Immunohistochemistry and real time RT-PCR.Result:1.The body weights were no significant difference between each group. At a certain dose range,the treatment group have the trend to shorten AGD following the dose increases.2.Exposure groups were observed testicular cord thin,cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.The results shows the level of testosterone step down in PND2 on male offspring.4.The results displaied the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.The immunohistochemistrical results show the protein expression of P450scc、3β-HSD,and PCNA are shortage(P＜0.05) in Leydig.the protein expression of AR is alike with control(P＞0.05).6.The results display the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR(P＜0.05),but the gene expression of AR identical with control.(P＞0.05).Conclusion:LIN can enter embryo and have adverse affects on F1 male rats:1.The body weights were no significant difference between each group.At a certain dose range, the treatment group have the trend to shorten AGD following the dose increases.2. Exposure groups were observed testicular cord thin、cells vacuolar degeneration and karyopyknosis increase compared with control group on pathological section.3.exposures to 50mg/kg LIN gestation might degrade the level of testosterone in PND2 on male offspring with ELISA.4.LIN displays the distension of rough endoplasmic reticulum and the swell of chondrosome in Leydig by electron microscope.5.LIN can degrade the protein expression of P450scc、3β-HSD and PCNA in Leydig.6.LIN cut down the gene expression of P450c17 degrade,the gene expression of 17β-HSD and PCNA weaken in Leydig in real time RT-PCR.更多还原