【作者】 姚丹；

【导师】 汝少国； 加藤秀生；

【作者基本信息】 中国海洋大学 ， 海洋生物， 2010， 博士

【摘要】 近年来,关于有机磷农药发育神经毒性的研究备受关注。久效磷作为一种高毒有机磷杀虫剂,由于具有水溶性特点,易在水环境中残留,对环境污染严重,甚至威胁着动植物乃至人类的健康。越来越多的研究表明有机磷农药对神经细胞复制和分化、轴突和树突的发生、乃至树突功能的影响都将导致行为上的缺陷。海胆的早期胚胎发育是研究农药胚胎和神经毒性的理想生物模型。因此,本文以马粪海胆胚胎和幼虫为实验材料,通过观察久效磷暴露条件下幼虫的形态发生和泳动行为,研究其对血清素和前多巴胺能系统调节功能的影响；采用整体免疫组化技术,从神经元形态发生的水平上,研究了久效磷对血清素顶系神经节及其受体细胞网络的抑制作用；并进一步探讨了血清素神经节异常情况下,轴突导向因子—HpNetrin的时空分布情况。主要研究结果如下：(1)形态学方面,久效磷会导致H.pulcherrimus幼虫腕长和体长的短小；运动行为方面,久效磷暴露条件下扰乱幼虫向上泳动的行为,甚至导致泳动能力的丧失,具有剂量-效应关系。(2)久效磷能够延迟早期神经发育进程,表现为减少血清素能神经细胞的数量和显著地抑制轴突的生长；血清素能神经元形态发生异常,特别是SRCN的重要组成部分口突横向纤维(oltort)的缺失,间接导致了血清素受体细胞网络(SRCN)的紊乱。(3)从10μg/ml久效磷暴露组开始HpNetrin在时间和空间上的表达异常,主要体现在棱柱幼虫时期动物极处无HpNetrin的富集,二腕幼虫时期沿纤毛带分布的HpNetrin信号较弱。采用免疫印迹技术对对照、10μg/ml和30μg/ml久效磷暴露组中HpNetrin蛋白从15-hpf到48-hpf阶段的表达量的半定量分析结果表明：久效磷扰乱了HpNetrin的表达模式,表现为15-hpf表达量较高,随后表达量逐渐降低,RT-PCR结果和免疫印迹结果相一致,说明久效磷从基因表达水平上扰乱了HpNetrin的表达。(4)0.1μg/ml久效磷暴露使绝大多数胚胎单细胞丧失重聚的能力,单细胞震动频率变小,但仍然存活；结合EpithⅡ免疫染色和台盼蓝染色法,我们发现久效磷对细胞膜具有较强的破坏作用；久效磷对重聚细胞团的毒性作用还表现在对血清素能神经元发生的影响。0.1μg/ml久效磷暴露后,仍然可以在重聚细胞团内检测到血清素细胞的阳性信号,但是无法检测到血清素受体细胞形成的神经纤维束结构,说明胚胎重组细胞模型可以用来检测血清素细胞的发生及其轴突的生长情况,而海胆幼体则更适合于研究久效磷对血清素受体细胞网络形成的影响。(5)久效磷暴露条件下,浮游囊胚期能够检测到DRD1阳性信号,但是与对照组相比颗粒数较少,而且分布不规则。通过Brdu免疫组化实验证明,48-hpf时延纤毛带分布的DRD1增殖数量显著降低,表明久效磷对多巴胺受体DRD1的时空表达具有抑制作用。更多还原

【Abstract】 Recently, more and more attention has been paid on the neurodevelopmental toxicity of organophosphorous pesticides. Monocrotophos is a kind of highly toxic organophosphorous pesticide. Due to its water-solubility, monocrotophos is apt to contaminate the environment with residue and even threaten the health of human’s. More and more researches demonstrated that the influence of organophosphorous on reproduction and differentiation of neurocyte, genesis of axon and dendrite and function of dendrite can cause defected behavior. Sea urchin embryo is an idealistic model for studying the toxicity of pesticide to embryo and nervous system. Therefore, in this study we use embryos and larvae of H. pulcherrimus as model to study the effects of monocrotophos on the formation and function of serotonergic and pre-dopaminergic nervous systems. As both serotonergic and pre-dopaminergic nervous systems have the function of regulate embryo and larvae’s swimming behavior. The morphology and swimming behavior of larvae were also observed here. The neurogenesis of serotonergic nervous system was illustrated by whole-mount immunohistochemistry and immunoblotting. Since the axon growth and serotonin receptor network formation were inhibited by monocrotophos. We also further studied spatio-temporal expression of HpNetrin in the sea urchin H. pulcherrimus during serotonergic axon extension. The mainly results are as follow:(1) Morphologically, MCP inhibited the body and arm growth of pluteus larvae; besides, larvae lost the ability of upright swimming, and this effect is dose dependant.(2) MCP delayed the early neurodevelopmental process of sea urchin. The results demonstrated that serotonergic cell decreased in number, and the axon growth of serotonin apical ganglion was significantly inhibited. The malformation of neuron morphogenesis also included the disorder of serotonin receptor network, especially the lost of oltort, which is one of the most important nerve fibers.(3) The spatio-temporal expression of HpNetrin was disordered from the concentration of 10μg/ml MCP. The whole-mount immunohistochemistry result showed that there is no increase intensity of HpNetrin protein near the animal pole of prism larvae, and very weak signal of HpNetrin along the ciliary band of 2-arm pluteus. The immunoblotting and RT-PCR results indicated that the amount of protein expression pattern was also disorded by MCP.(4) Dissociated cells lost the ability to re-aggregate under the exposure of 0.1μg/ml MCP. Single cells shaked weakly, but still alive; Epith II immuno stain and Trypan Blue stain results showed that the cell membrane were destroyed by MCP; The neurogenesis of serotonergic neurons were also affect in re-aggregated cells, which is correspondence to the results obtain by study on intact organism. But the effect of MCP on the formation of serotonin receptor network formation can’t be detected in re-aggregated cells. Thus, although re-aggregated cell is more sensitive than larvae, it still has limitations in the screening of neuro-toxicants.(5) The positive signal of dopamine receptor D1 (DRD1) can be detected since the swimming blastula stage of sea urchin. Our results illustrated that the DRD1 granules decrease obviously with the incubation of 10μg/ml MCP. We proved that reproduction of DRD1 along the ciliary band of 48-hpf larvae reduced greatly in number by BrdU incorporation experiment.更多还原