微囊藻毒素长期暴露对青少年学习记忆的影响及分子机制研究

The pollution of microsystins(MCs）in water is more and more serious .The previous research reported that MCs can damage learning and memory ability, but the mechanism is not clear. We revealed that the level of MCs in source water in some towns of Three Gorges Reservoir area is very high. The children's daily intake of MC-LR(one kind of typical MCs) were 5 times of WHO standard, and the MC-LR in serum could be detected in the rate of 91.9%. Since child is more sensitive to toxicant than adult, study the effect and mechanism of MC-LR on learning and memory of children are very important. After analyzed the data in literatures and our research，we presume that MCs probably influence the N-methyl-D-aspartic acid receptor (NMDAR) induced long-term potentiation (LTP) according to disturb the key molecule CaMKⅡ、kalirin-7 , then damage learning and memory. In this study, we will investigate the current situation of damage of learning and memory of 1132 children induced by MC-LR, and explore the mechanism of it in rat and primary neuron cell by the method of siRNA, western-blot and other molecular biology technique. Our aim is to provide data for prevent the hazard of MCs on children and find molecular targets for intervention.

水环境污染和气候变暖导致水中微囊藻毒素（microsystins,MCs）污染严重。既往研究表明MCs损害学习记忆能力，但机制不清。课题组前期发现重庆市三峡库区某现场水源微囊藻毒素污染严重，儿童MC-LR(MCs典型代表)日摄入量为WHO规定限值5倍，血中MC-LR检出率91.9%。青少年对毒作用较成人更敏感，研究MCs长期暴露对其学习记忆能力的影响及毒作用机制尤为迫切。结合文献及前期实验结果我们推测MCs入脑后可能通过抑制蛋白磷酸酶PP1干扰学习记忆关键分子CaMKⅡ、kalirin-7等而扰乱NMDAR介导的长时程增强（LTP），从而损伤学习记忆能力。本项目拟在该现场已建立的1132人MCs暴露青少年队列人群中调查MC-LR对学习记忆能力影响现况，并用免疫印迹、RNA干扰等技术从整体动物、细胞、分子水平探讨其机制，为防治MCs暴露对青少年的危害提供资料，并在分子与细胞水平寻找干预靶点。

饮水中微囊藻毒素（Microcystin, MC）污染已成为威胁人们饮用水安全主要因素之一。本项目在三峡库区某MC污染较为严重地区青少年（1043人）中进行考试成绩资料收集、问卷调查、血液采集、数字符号测验、数字划消实验、瑞文智力测验、韦氏智力测试等。检测了饮水及水产品MC-LR及血液MC-LR和铅含量，分析其与学习成绩和智力、记忆力和神经功能测试结果的关系。并MC-LR染毒SD大鼠和神经细胞PC12探索其对学习记忆能力影响机制。.纳入697名青少年血中MC-LR中位数为0.80 ng/mL，血铅为44.31 μg /L。0.80-0.95μg /L MC-LR暴露组（Q3）学生的学习成绩、数字符号实验(DSST)、记忆商数（MQ）、短时记忆得分（STM）、长时记忆得分（LTM）均显著高于Q1。Q4血铅组（>63.31µg/L）LTM 得分显著降低，高MC-LR组(≥0.80μg /L)和高血铅组(≥44.31μg /L)相比低血铅(<44.31μg /L)+低MC-LR(<0.80μg /L) MQ 得分与 LTM 得分显著增加，MC-LR与铅之间有一定交互作用。.1μg/kg bwMC-LR染毒10周后大鼠海马Kalirin-7基因表达有上升趋势，kalirin和PP1在CA1和CA3区锥体细胞、DG颗粒细胞中表达增加，高剂量组大鼠海马神经元细胞出现线粒体和内质网水肿、暗细胞数量增加等损伤， kalirin-7基因表达出现下降趋势且PP1在CA1和CA3区锥体细胞、DG颗粒细胞中表达下降。CaMKⅡ、NMDAR1和NMDAR2B蛋白表达差异无统计学意义。MC-LR 染毒剂量增加使PC12细胞形态变圆变小，突触变短减少，神经丝连接不明显，100、200μg/L MC-LR 组Kalirin-7与NMDR2B基因表达显著降低；50、100、200μg/L MC-LR 组NMDR2B蛋白和PSD-95基因均显著降低。.综上所述，血清MC-LR水平在0.80-0.95μg /L之间的青少年学习记忆能力有所增强；MC-LR在低剂量时可诱导大鼠海马PP1和Kalirin 的表达增加可能是其潜在的机制。但高剂量的MC-LR可损伤大鼠海马超微结构，改变PC12细胞的形态并降低Kalirin-7和PSD-95的表达。该结果将为防治微囊藻毒素对青少年学习记忆能力提供依据。