【作者】 周晓云；

【导师】 谭凤珠；

【作者基本信息】 河北医科大学 ， 劳动卫生与环境卫生学， 2008， 硕士

【摘要】 目的2 , 3 , 7 , 8 -四氯二苯-对-二恶英( 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin,TCDD)是地球上毒性最强的化合物之一,国际癌症研究中心(International Agency for Research on Cancer,IARC)将其列为Ⅰ级致癌物。世界卫生组织(WHO)等机构认为,虽然TCDD是强致癌物,但就目前其暴露背景而言,TCDD的非致癌毒性作用比致癌毒性作用对人体健康危害的风险更大。此外,TCDD具有结构稳定、半衰期长的特性,仅需暴露一次就可以长期留存体内,长期接触可造成体内蓄积,造成严重的机体毒害作用。由于幼鼠对TCDD高度敏感,所以本研究在雌性小鼠分娩后立即经腹腔注射低剂量TCDD,通过乳汁对仔鼠染毒,然后观察哺乳期暴露于TCDD对仔鼠体重增长、神经发育和学习记忆的影响,大脑海马和皮层组织结构以及细胞色素P450 1A1酶(cytochrome P450 1A1,CYP1A1)、凋亡基因Bcl-2(B-Cell lymphoma/leukemia 2)和Bax(Bcl-associated x protein)蛋白表达量的变化,为低剂量TCDD造成的子代慢性神经系统危害及其机制提供科学依据。方法1动物模型建立与分组4月龄健康成年昆明小鼠,雌鼠24只,雄鼠8只,按照3:1的雌雄比例同笼受孕。分娩7~10只仔鼠的保留,调整每窝仔鼠8只,雌雄对半,其余淘汰。将母鼠和仔鼠作为整体(窝)进行完全随机分组,设2个试验组:40μg和20μg TCDD/kg体重组;与试验组对应,设2个溶剂对照组:40μg和20μg溶剂对照组;另设1个不做任何处理的空白对照组;共5组,每组3窝(5个试验组以下简写为40μg TCDD组、20μg TCDD组、40μg溶剂对照组、20μg溶剂对照组和空白对照组)。雌鼠分娩之后,立即称重并进行腹腔注射。母鼠哺乳仔鼠(即染毒)至仔鼠出生后第21天,而后停止哺乳,让仔鼠自由进食。仔鼠出生后第35天被处死。TCDD浓度为10μg/ml(TCDD/甲苯),纯度为99%,腹腔注射前将该试剂用花生油稀释,体积比为TCDD:花生油=1:4,则浓度稀释为2μg/ml。溶剂对照为甲苯和花生油按1:4体积比混合的混合液。2仔鼠体重的测定3仔鼠神经发育指标的测定(平面翻正试验、负趋地试验、前肢握力试验、空中翻正试验、足展开试验)4仔鼠学习记忆能力的测定(Y迷宫试验)5采用免疫组织化学染色方法对仔鼠大脑海马和皮层组织CYP1A1蛋白进行定位和半定量分析。6仔鼠大脑海马和皮层组织细胞凋亡的测定6.1采用H.E染色法观察仔鼠大脑海马和皮层组织的结构变化6.2采用免疫组织化学染色法对仔鼠大脑海马和皮层组织Bcl-2和Bax凋亡蛋白进行定位和半定量分析。6.3采用流式细胞术对仔鼠大脑海马和皮层组织Bcl-2和Bax蛋白表达量进行定量分析结果1 TCDD对仔鼠体重的影响新生仔鼠体重基本相同(P>0.05);出生后第7天,20μg TCDD组仔鼠的平均体重低于其溶剂对照组和空白对照组(P<0.01);随着TCDD的摄入和蓄积,40μg和20μg TCDD组仔鼠的平均体重均低于相应的溶剂对照组和空白对照组(P<0.01),并且40μg TCDD组仔鼠的平均体重低于20μg TCDD组(P<0.05);停止染毒后(出生后第28天和第35天),暴露于TCDD仔鼠的平均体重依然低于相应的溶剂对照组和空白对照组(P<0.01)。2 TCDD对仔鼠神经发育的影响哺乳期暴露于TCDD的仔鼠的平面翻正和负趋地时间大于相应的溶剂对照组和空白对照组,前肢握力时间和足展开距离小于相应的溶剂对照组和空白对照组,空中翻正正确率低于相应的溶剂对照组和空白对照组,具有统计学差异(P<0.01)。3 TCDD对仔鼠学习记忆的影响在Y迷宫测试中,哺乳期暴露于TCDD的仔鼠获取记忆的能力(A)、保持记忆的能力(B)和记忆保持率(C)均低于相应的溶剂对照组和空白对照组,其差别具有统计学意义(P<0.01)。4 TCDD对仔鼠大脑海马和皮层组织CYP1A1蛋白表达量的影响CYP1A1蛋白主要表达于仔鼠大脑海马和皮层组织锥体细胞的胞浆中。哺乳期低剂量暴露于TCDD可使仔鼠大脑锥体细胞CYP1A1蛋白的表达量增高(P<0.01),各组雌性仔鼠的CYP1A1蛋白表达量略高于雄性仔鼠,但差异无统计学意义(P>0.05)。5 TCDD对仔鼠大脑海马和皮层组织细胞凋亡的影响哺乳期低剂量暴露于TCDD的仔鼠的大脑海马和皮层组织锥体细胞排列紊乱,胞体皱缩,核染色质浓集,呈现细胞凋亡现象。凋亡基因Bcl-2主要表达于仔鼠大脑锥体细胞的胞浆中,并且存在性别差异,雌性仔鼠高于雄性(P<0.01)。哺乳期低剂量暴露于TCDD可使雌雄性仔鼠大脑锥体细胞Bcl-2蛋白的表达量减低(P<0.01),但雌性仔鼠Bcl-2蛋白表达量始终高于雄性(P<0.01)。凋亡基因Bax主要表达于仔鼠大脑锥体细胞的胞浆和胞核中,表达量不存在性别差异(P>0.05)。哺乳期低剂量暴露于TCDD可使雌雄性仔鼠大脑锥体细胞Bax蛋白的表达量增高(P<0.01),并且雌性仔鼠增高较雄性明显,存在性别差异(P<0.05)。TCDD可以使雌雄仔鼠大脑锥体细胞Bcl-2/Bax比值降低(P<0.01),说明TCDD可以诱导雌雄仔鼠海马和皮层组织锥体细胞凋亡。并且雄性仔鼠Bcl-2/Bax比值较雌性低,差异具有统计学意义(P<0.01),说明雄性仔鼠比雌性的细胞凋亡率高。结论1哺乳期低剂量暴露于TCDD,可以阻碍仔鼠的体重增长,并且这种阻碍作用并不随着染毒的停止而停止,具有持续性。2哺乳期低剂量暴露于TCDD,可以使仔鼠的神经反射功能下降,运动协调能力降低,肌张力减退,神经发育迟缓。并且接触量越大,蓄积量越多,对神经发育的影响越明显。3哺乳期低剂量暴露于TCDD,可以使仔鼠的学习记忆能力下降,危险回避能力受损。4哺乳期低剂量暴露于TCDD,可以使仔鼠大脑海马和皮层组织CYP1A1蛋白表达量增加,说明血脑屏障对TCDD的阻隔作用有限,TCDD可以通过尚未发育好的血脑屏障进入脑组织,造成海马和皮层组织锥体细胞的损伤,这可能是TCDD导致子代神经发育迟缓,学习记忆能力下降的重要途径之一。5哺乳期低剂量暴露于TCDD,可以诱导仔鼠大脑海马和皮层组织锥体细胞呈现凋亡改变,可以使Bcl-2蛋白表达量减低,Bax蛋白表达量增高,Bcl-2/Bax比值降低。Bcl-2凋亡基因家族比值的改变可能是TCDD导致细胞异常凋亡的重要机制之一,也可能是TCDD导致子代神经发育迟缓,学习记忆能力下降的途径之一。并且Bcl-2/Bax比值的雌雄差异,可能是雌雄仔鼠对TCDD敏感度不同、损害程度不同的原因之一。更多还原

【Abstract】 ObjectiveThe contaminant 2, 3, 7, 8-tetrachlordibenzo-p-dioxin (TCDD) is one of the most toxical compounds and ranked asⅠlevel carcinogen by International Agency for Research on Cancer (IARC). It is considered by many agencies such as WHO that TCDD belongs to the category of high carcinogenicity, but the risk of non-carcinogenicity toxic effect on human health is higher than that of carcinogenicity toxic effect in present exposure background. In addition, structural stability and long half-life period lead to TCDD staying or accumulating in human body after once or long-time exposure. As a result, TCDD induce severe toxic effects to the organism.For mice offspring are hypersensitive to TCDD, in this study, the female mice got TCDD by intraperitoneal injection immediately after parturition and the offspring got by breast feeding. The present study would identify the toxic effect of lactational exposure to low level TCDD on mice offspring by observing body weight, neural development, learning and memory ability, the histological changes and expression of cytochrome P450 1A1 enzyme (CYP1A1), apoptosis gene B-Cell lymphoma/leukemia 2 (Bcl-2) and Bcl-associated x protein (Bax) in hippocampus and cerebral cortex tissue. The results would provide evidence for the study about the chronic nervous system effect and mechanism of lactational exposure to low level TCDD on mice offspring.Methods1 Animals model and groups4-month-old mature Kunming mice with 24 female and 8 male were grouped by 3:1. After parturition, the female mice with 7~10 pups were retained, and 8 pups were adjusted as a unit, including 4 female and 4 male offspring. The others were eliminated. Then the female mice and its offspring, regarded as a unit, were divided into 5 groups randomly. In this study, there were 2 doses of treatment: 40μg and 20μg TCDD/kg body weight. Corresponding to 2 doses of TCDD treatment, there were 2 vehicle controls: 40μg and 20μg vehicle/kg body weight, and 1 animal control without any treatment. There were 5 groups totally and each group had 3 units (They noted as 40μg TCDD, 20μg TCDD, 40μg vehicle control, 20μg vehicle control and animal control group respectively). After parturition, the female mice were weighted and intraperitoneally injected. The female mice stop lactation on offspring postnatal days (PND) 21, and then offspring were fed on animal feeds. Mice offspring were killed on PND 35.TCDD was dissolved in methylbenzene with a concentration of 10μg/ml and a purity of 99% when purchased, and diluted with peanutoil by 1:4. The control vehicle was the mixed liquor with methylbenzene and peanutoil by 1:4.2 Measurement of mice offspring’s body weight.3 Measurement of mice offspring’s neurodevelopment indexes (surface fighting, negative geotaxis, forelimb grip force, air fighting, hindlimb splaying).4 Measurement of mice offspring’s learning and memory ability (Y-maze test).5 Localization and semi-quantitative analysis of CYP1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspring by immunohistochemistry.6 Detection of apoptosis in hippocampus and cerebral cortex tissue of mice offspring.6.1 Observation of structure changes in hippocampus and cerebral cortex tissue of mice offspring by H.E staining method.6.2 Localization and semi-quantitative analysis of Bcl-2 and Bax protein expression in hippocampus and cerebral cortex tissue of mice offspring by immunohistochemistry.6.3 Quantitative analysis of Bcl-2 and Bax protein expression in hippocampus and cerebral cortex tissue of mice offspring by flow cytometry.Results1 The effect of TCDD on the body weight of mice offspring There was no difference in the average body weight of mice offspring between 5 groups when the offspring were born (P>0.05). On PND 7, the average body weight in 20μg TCDD group decreased compared with 20μg vehicle control and animal control groups (P<0.01). Along with the time of breast feeding, the average body weight in 40μg and 20μg TCDD groups decreased compared respectively with vehicle control and animal control groups (P<0.01), and that in 40μg TCDD group also decreased compared with 20μg TCDD group (P<0.05). After mice offspring were weaned (on PND 28 and PND 35), the average body weight in two TCDD groups also decreased compared with vehicle control and animal control groups (P<0.01).2 The effect of TCDD on the neurodevelopment of mice offspringThe time of surface fighting and negative geotaxis in two TCDD groups were longer than those in vehicle control and animal control groups (P<0.01). The time of forelimb grip force and the distance of hindlimb splaying in two TCDD groups were shorter than those in vehicle control and animal control groups (P<0.01). The correct rates of air fighting in two TCDD groups also decreased compared respectively with vehicle control and animal control groups (P<0.01).3 The effect of TCDD on learning and memory ability of mice offspringIn Y-maze test, the abilities of learning (A), memory (B) and the memory retaining rate (C) were decreased in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). 4 The effect of TCDD on CYP1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspringCYP1A1 protein expressed mainly in the cytochylema of pyramidal cell in hippocampus and cerebral cortex tissue of mice offspring. The average optical density of CYP1A1 protein increased in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). The female offspring’s CYP1A1 protein expression was higher than the male offspring’s, but there was no statistical significance of the difference (P>0.05).5 The effect of TCDD on apoptosis in hippocampus and cerebral cortex tissue of mice offspringLactational exposure to low level TCDD led to pyramidal cell apoptosis such as disordered structure, cell body crenation and chromatin agglutination in hippocampus and cerebral cortex tissue of mice offspring.Bcl-2 protein expressed mainly in the cytochylema of pyramidal cell in hippocampus and cerebral cortex tissue of mice offspring. It had statistical difference between the female and male offspring in vehicle control and animal control groups, and the average optical density of Bcl-2 protein of female offspring was higher than that of male offspring (P<0.01). Lactational exposure to low level TCDD made Bcl-2 protein expression decreased both in female and male offspring(P<0.01), but the female offspring’s Bcl-2 protein expression was still higher than the male offspring’s (P<0.01). Bax protein expressed mainly in pyramidal cell membrane and cytochylema in hippocampus and cerebral cortex tissue of mice offspring. It had no statistical difference between the female and male offspring in vehicle control and animal control groups (P>0.05). Lactational exposure to low level TCDD made Bax protein expression increased both in female and male offspring(P<0.01), and the female offspring’s Bax protein expression was higher than the male offspring’s (P<0.05).The ratio of Bcl-2/Bax decreased significantly in two TCDD groups compared respectively with vehicle control and animal control groups (P<0.01). It suggested that TCDD would induce pyramidal cell apoptosis in hippocampus and cerebral cortex tissue of mice offspring. Furthermore, the Bcl-2/Bax ratio of male offspring was lower than that of female offspring (P<0.01). It suggested that the apoptosis rate of male offspring was higher than that of female.Conclusion1 Lactational exposure to low level TCDD would delay the body weight gain of mice offspring. This adverse effect was persistent and did not vanish when stopping TCDD intake.2 Lactational exposure to low level TCDD would step down the function of nervous reflex, decrease the ability of movement and coordination, degrade the tension of muscle and delay the development of nervous system of mice offspring. And the more dose TCDD intook, the more obvious the adverse effect was.3 Lactational exposure to low level TCDD would impair the spatial learning and memory abilities and damage the danger-avoiding capability of mice offspring.4 Lactational exposure to low level TCDD would increase CPY1A1 protein expression in hippocampus and cerebral cortex tissue of mice offspring. It demonstrated that blood-brain barrier had a limited effect on preventing TCDD from entering the brain tissue, so TCDD could damage hippocampus and cerebral cortex tissue. It might be one of the important mechanisms of TCDD delaying neural development and impairing learning and memory ability of mice offspring.5 Lactational exposure to low level TCDD would induce pyramidal cell apoptosis, decrease Bcl-2 protein expression, increase Bax protein expression, decrease the Bcl-2/Bax ratio in hippocampus and cerebral cortex tissue of mice offspring. The ratio changes of Bcl-2 apoptosis gene family might be one of the important mechanisms of TCDD inducing abnormal apoptosis, and might also be one of the mechanisms for TCDD delaying neural development and impairing learning and memory ability. In addition, the sexual difference in the Bcl-2/Bax ratio in hippocampus and cerebral cortex tissue might be one of the causes why there were significant discrepancies in TCDD sensitivity and impairment between the female and male offspring.更多还原