【作者】 李秀娟；

【导师】 蒋莉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2011， 博士

【摘要】 第一部分围生期DBP暴露对不同发育期子代大鼠海马神经细胞的影响目的：在本课题组前期研究已证实500mg/（kg.d）DBP围生期暴露对子代大鼠的学习记忆有影响的基础上，进一步观察该剂量DBP围生期暴露对不同发育期子代大鼠海马神经细胞的影响，并探讨其是否具有年龄、性别差异性，以及发育早期停止暴露后DBP对成年仔鼠海马神经细胞是否仍然有影响。方法：SD孕鼠从孕第6天至产后21天给予500mg/（kg.d）DBP或同等容积玉米油（正常对照组）灌胃染毒，仔鼠21天断乳后正常喂养，分别于仔鼠生后5天、21天、60天取海马检测以下指标：①尼氏染色观察海马神经细胞存活情况；②透视电镜观察海马神经细胞超微结构；③原位末端标记法（TUNEL）检测海马凋亡细胞；④Annexin V-PI双参数流式细胞法检测海马神经细胞死亡和凋亡情况；⑤酶标仪法检测海马神经细胞Caspase-3活性。结果：①尼氏染色：给药组5天及21天仔鼠海马各区神经细胞排列紊乱、细胞轮廓不清或溶解，神经细胞数较正常对照组明显减少，雌雄无差异；与正常对照组仔鼠比较，给药组5天雌雄仔鼠神经细胞减少比例分别高达13.4%和15.3%，给药组21天雌雄仔鼠神经细胞减少比值分别为7.9%和4.7%，表明DBP围生期暴露导致海马神经细胞丢失，且新生鼠较幼年鼠更明显；而给药组60天仔鼠与对照组比较虽然有所降低，但无统计学差异。②透视电镜：给药组21天的雌、雄仔鼠海马神经细胞均出现线粒体肿胀、空泡化、内质网扩张改变；给药组60天的雌、雄仔鼠神经细胞超微结构未见明显异常改变。③TUNEL法检测海马细胞凋亡：给药组5天、21天仔鼠海马凋亡细胞较对照组明显增多，而给药组60天仔鼠与对照组比较无显著差异，雌雄仔鼠间凋亡细胞无差异。④Annexin V-PI法检测海马神经细胞死亡和凋亡情况：给药组5天雌雄仔鼠海马死亡细胞比例均较对照组增高，P＜0.01；给药组5天、21天仔鼠海马凋亡细胞比例较对照组明显增高，雌雄无差异，P均＜0.001。与同龄同性别正常对照比较，给药组5天雌雄仔鼠凋亡细胞增加比值分别高达278.4%和376.4%，而给药组21天雌雄仔鼠增加比值分别为169.8%和159.1%，表明DBP围生期暴露导致的海马细胞过度凋亡新生鼠较幼年鼠更明显。给药组60天仔鼠海马凋亡细胞比例与对照组比较有增高，但无统计学差异。⑤海马神经细胞Caspase-3活性检测：给药组5天、21天仔鼠海马Caspase-3活性均较对照组明显增高，雌雄间无差异；与同龄同性别正常对照比较，给药组5天雌雄仔鼠海马细胞caspase-3活性增加比值分别高达130.1%和166.1%，而给药组21天雌雄仔鼠增加比值分别为39.6%和38.2%，再次表明DBP围生期暴露导致的海马细胞过度凋亡新生鼠较幼年鼠更明显；给药组60天仔鼠与对照组比较无显著差异。结论：①500mg/(kg·d)DBP围生期染毒可导致新生鼠和幼年鼠海马神经细胞坏死和过度凋亡，年龄越小，损害越重，这可能是DBP导致发育期大鼠学习记忆功能障碍的原因之一。②DBP对海马神经细胞的毒性效应未见明显性别差异。③发育早期脱毒后，DBP未对成年脑海马神经细胞造成明显损伤。第二部分围生期DBP暴露对未成熟期子代大鼠海马突触的影响目的：观察DBP围生期暴露对未成熟期子代大鼠海马突触结构和功能的影响，并分析性别差异性及发育早期脱毒后的变化。方法：模型建立及分组方法同第一部分，分别于仔鼠生后21天、60天取海马进行检测：①免疫组化及Western blot方法分析子代大鼠海马突触素的表达变化。②电镜观察海马突触超微结构。③观察海马突触CA1区长时程增强的变化。结果：①海马突触素表达：正常对照组60天雌雄仔鼠的海马突触素表达较21天同性别仔鼠明显增高，P均＜0.001；给药组21天仔鼠海马突触素表达较对照组明显降低，雌雄无差异，P均＜0.01；而给药组60天仔鼠突触素表达与对照组比较虽有降低，但无统计学差异。②电镜观察海马超微结构：与对照组比较，给药组21天雌雄仔鼠海马突触均呈现出数量减少、突触前囊泡减少、突触后致密物变薄的变化趋势，而给药组60天仔鼠海马突触无明显类似改变。③海马CA1区锥体细胞突触长时程增强的变化：无论雌雄，HFS后正常对照组60天仔鼠fEPSP的斜率和幅度变化均较正常对照组21天仔鼠增高，P均＜0.05；给药组21天雌雄仔鼠HFS后的fEPSP斜率及幅度变化均较对照组显著降低；而给药组60天仔鼠HFS后的fEPSP的斜率和幅度变化与正常对照组比较同样有所下降，但仍然无统计学差异。各组间雌雄无差异。结论：①500mg/(kg·d)DBP围生期暴露可明显损害幼年子代大鼠海马突触结构和突触传递效能，无性别差异性，这可能是DBP导致发育期子代大鼠学习记忆功能障碍的另一个重要原因。②500mg/(kg·d)DBP围生期暴露仔鼠成年后的海马突触结构及突触传递效能虽有轻微异常，但与正常对照比较没有统计学意义，提示发育早期脱离污染后，DBP对成年脑海马突触没有明显影响。第三部分围生期DBP暴露致子代大鼠神经毒性的可能机制目的：观察DBP暴露后子代大鼠的血清性激素水平变化、海马AROM及ER-β的表达水平，以及与ER-β信号途经相关的两个重要蛋白p-CREB、BDNF的表达变化，探讨DBP神经毒性可能机制。方法：模型建立及分组方法同第一部分，分别于仔鼠生后21天、60天取血清和海马进行检测：①化学发光酶联免疫法检测子代大鼠血清中的睾酮和雌二醇水平；②采用免疫组化及Western blot方法分析子代大鼠海马AROM、ER-β、p-CREB、BDNF的表达变化。结果：①血清性激素水平变化：给药组21天、60天雄鼠及21天雌鼠血清睾酮水平较同龄对照组低，而21天、60天雌雄大鼠雌激素水平均较对照组增高。②海马AROM、ER-β、p-CREB、BDNF表达的变化：与对照组仔鼠比较，给药组21天仔鼠的AROM的表达增高，而ER-β、p-CREB、BDNF的表达均降低，雌雄无显著差异；而给药组60天仔鼠各指标与对照组比较无显著差异。结论：①DBP围生期暴露降低了雌雄子代大鼠的睾酮水平，且雄性子代大鼠的睾酮降低持续至成年期；而幼年雌雄子代大鼠血清雌激素水平均增高，一直持续至成年。②DBP围生期暴露上调了幼年子代大鼠海马AROM表达，而抑制了ER-β、p-CREB及BDNF的表达。血清睾酮水平的降低及海马脑区ER-β的表达下调导致p-CREB依赖性BDNF雌激素效应的降低，可能是DBP导致海马神经细胞毒性及突触可塑性损害的关键原因。③DBP对海马AROM、ER-β、p-CREB及BDNF表达的影响均未见性别差异，并在大鼠成年后基本恢复正常，提示发育早期脱离DBP暴露能够避免持续性神经毒性损伤。更多还原

【Abstract】 PART Ⅰ EFFECTS OF DIBUTYL PHTHALATE ONHIPPOCAMPAL NEURONS IN RATS OF DIFFERENTDEVELOPMENTAL STAGES FOLLOWING PERINATAL EXPOSUREObjective: To observe the effects of Dibutyl Phthalate (DBP)at dose of500mg/(kg.d) on hippocampal neurons in rat ofdifferent developmental stages following perinatal exposure,and to investigate whether the effects have age and genderdifferences，finally to find out whether the neurotoxicity ofDBP is still significant for adulthood offspring rats aftercessation of exposure in early developmental stages.Methods: Pregnant Sprague Dawley rats were gavaged dailywith500mg/(kg.d) DBP or with corn oil (control) from gestationday (GD)6to postnatal day (PND)21and the Offspring rats werefed normally after weaning on PND21, then the hippocampus ofoffspring rats were obtained on PND5、PND21and PND60and the following will be carried out:①Counting surviving neurons andobserving morphology changes by Nissl staining;②Observingultrastructural changes of neurons using transmission electronmicroscopy;③Detecting neuronal apoptosis and necrosis byTUNEL and by flow cytometry with Annexin V-PI dual parameters;④Detecting caspase-3activity by Microplate Reader.Results:①Neurons in hippocamps disordered and the nisslbody degraded in DBP-treated group on PND5and PND21, neuronalnumber was also significantly decreased compared with controlgroup，no significant difference was found between male andfemale rats. Moreover, compared with control, neuronal numberwas reduced by13.4%and15.3%in female and male offspringsrespectively on PND5, while was reduced by7.9%and4.7%in femaleand male offsprings on PND21. Neuronal number in DBP-treated ratson PND60also decreased compared with control, but withoutsignificant difference.②Ultrastructure of hippocampalneurons showed mitochondria swelling, even vacuolization andendocytoplasmic reticulum dilatation in male and femaleDBP-treated rats on PND21, while none obvious pathologic changeswere detected in DBP-treated rats on PND60and in control rats.③Compared with control, significant increasing neoronalapoptosis were found in male and female DBP-treated rats on PND5 and PND21both by TUNEL assay and flow cytometry assay; Comparedwith DBP-treated rats on PND21, a more significant increasingneuronal apoptosis was found in DBP-treated rats on PND5.Although neuronal apoptosis in DBP-treated rats on PND60increased compared with control, but there was no significantlydifference.④Both male or female rats in DBP-treated group onPND5and PND21,caspase-3activity were significantly increasedwhen compared to control, and the increasing of caspase-3activity in DBP-treated rats on PND5was more apparent than ratson PND21.Similar with the results above, no significantdifference was observed between DBP-treated rats on PND60andrats in control group on PND60.Conclusion:①Perinatal exposure to500mg/(kg.d) DBP mayincrease neuronal death and apoptosis in hippocampus of bothnewborn and childhood offspring rats without gender differences.And the damage was more severe in newborn rats than in childhoodrats.②DBP failed to cause adulthood hippocampal neurons damagesignificantly after cessation of exposure in early stages ofdevelopment. PART Ⅱ EFFECTS OF DIBUTYL PHTHALATE ONHIPPOCAMPAL SYNAPSES OF IMMATURE OFFSPRING RATSFOLLOWINGPERINATA EXPOSUREOBJECTIVES: To investigate the effects of DBP on hippocampalsynapses of immature offspring rats following perinatal exposure,and to clarify whether the effects have gender differences,whether the effects are still significant for adulthoodoffspring rats after cessation of exposure in early stages ofdevelopment.METHODS: Pregnant Sprague Dawley rats were gavaged dailywith500mg/(kg.d) DBP or with corn oil (control) from gestationday (GD)6to postnatal day (PND)21and the Offspring rats werefed normally after weaning on PND21, then synaptophysin levelin hippocampus were detected by immunohistochemistry and westernblot, ultrastructural changes of hippocampal synapses wereobserved using transmission electron microscopy, and fieldexcitatory postsynaptic potentials (fEPSPs) in CA1area ofhippocampal slices after high-frequency stimulation(HFS) wereanalyzed with patch clamp when offspring rats were on PND21andPND60. RESULTS:①Synaptophysin level decreased markly inhippocampus in both male and female DBP-treated pups on PND21compared with the control (P<0.01), though synaptophysin levelalso decreased in both male and female DBP-treated pups on PND60,but the difference was not statistically significant comparedwith control.②Ultrastructure of hippocampal synapses showedthe number of synapses reduced, synaptic vesicles decreased andpostsynaptic density got thinner in both male and femaleDBP-treated pups on PND21compared with the control, but nonesimilar abnormal changes were detected in DBP-treated rats onPND60.③The changing degrees of fEPSPs slope and amplitudeafter HFS were significantly lower in both male and femaleDBP-treated pups on PND21than the control. However, all the datain DBP-treated rats on PND60decreased too but withoutsignificant difference compared with the ras in control group.CONCLUSIONS:①Perinatal exposure to500mg/(kg.d) DBP cansignificantly damage hippocampal synapse structure andeffectiveness of synaptic transmission in DBP-treated childhoodrat without gender differences.②The impact of DBP perinatalexposure on synapse structure and function in adult offspringrats was minor, and was not statistically significant comparedwith rats in control group, which showed DBP also failed to cause adulthood hippocampal synapse damage significantly aftercessation of exposure in early developmental stages. PART Ⅲ POSSIBLE MECHANISMS OF DBP NEUROTOXICITYON OFFSPRING RATS FOLLOWING PERINATAL EXPOSUREOBJECTIVES:To observe sex hormone levels in serum and AROM、ER-β、p-CREB and BDNF levels in hippocampus of offspring ratsfollowing perinatal DBP exposure, and to probe possiblemechanisms of neurotoxicity of DBP.METHODS: Drug administration and animal grouping in thispart are the same as that in part I and II, then the serum andhippocampus of off-spring rats were obtained on PND21and PND60for testing thefollowings:①Testosterone and estradiol levels in serum bychemiluminescent enzyme-linked immunosorbent assay;②AROM,ER-β, p-CREB and BDNF levels in hippocampus of offspring ratsby immune-histochemistry and Western blot.RESULTS:①Sex hormone levels in Serum: Compared with thecontrol, testosterone levels in serum significantly decreasedin male DBP-treated pups on PND21and PND60, testosterone level also decreased significantly in female DBP-treated pups on PND21;estradiol levels in serum significantly increased for both maleand female DBP-treated pups on PND21and PND60.②AROM, ER-β,p-CREB, BDNF levels: The AROM level increased apparently and thelevels of ER-β, p-CREB and BDNF decreased significantly inhippocampus in both male and female DBP-treated pups on PND21compared with the control, but no significant differences weredetected in DBP-treated rats on PND60compared with rats incontrol group. And there was no significant difference betweenmale and female pups in all groups.CONCLUSIONS:①Perinatal DBP exposure reduced testosteronelevels in serum in both male and female childhood offspring ratsand the reducing of testosterone level continued till adulthoodin male offspring rats. While perinatal DBP exposure increasedestradiol levels in serum in childhood and adulthood offspringrats in both genders.②DBP increased AROM level and inhibitedER-β、p-CREB and BDNF expression in hippocampus of childhoodoffspring rats. The reducing of serum testosterone and ER-βexpression in hippocampus led to the decreasing ofp-CREB-dependent BDNF level, which may be the key reason for theneurotoxic effect caused by DBP.③The changing levels of AROM、ERβ、p-CREB and BDNF in hippocampus after DBP expore were without gender differences, and returned to normal in adult rats,suggesting cessation of exposure in early developmental stagescan avoid persistent neurotoxic damage caused by DBP.更多还原