铅暴露对血脑脊液屏障维持铜稳态的影响及机制研究
发布时间：2018-07-25 14:06
【摘要】：目的本研究通过建立铅暴露大鼠模型,检测铅暴露大鼠中枢神经系统海马、皮质、脉络丛以及血液和脑脊液中铜含量变化;铅暴露对血脑脊液屏障铜清除能力的影响,进而从脉络丛铜转运蛋白(CTR1、ATP7A)及伴侣蛋白(COX17)角度初步探讨铅对中枢神经系统铜蓄积的机制。本研究结果将进一步丰富铅神经毒性的机制,同时也为铅神经毒性的标志物研究提供重要信息及相关靶点。方法45只健康雄性SD大鼠随机分为对照组、300ppm铅暴露组、600ppm铅暴露组。适应性饲养7天后,通过自由饮水的染毒方式给予铅暴露组大鼠300ppm、600ppm的醋酸铅饮用水,对照组大鼠给予600ppm的醋酸钠饮用水,染毒时间为24周。每日观察记录大鼠精神状态、活动量、饮食及饮水等情况,每周检测大鼠体重变化。染毒24周结束后,应用Morris水迷宫和旷场实验检测大鼠的神经功能;应用ICP-MS检测大鼠全血、皮质、海马、脉络丛及脑脊液中铅、铜含量;利用侧脑室灌注方法检测铅暴露对大鼠血脑脊液屏障铜清除能力的影响;应用激光扫描共聚焦显微镜观察脉络丛上皮细胞铜相关蛋白(ATP7A、CTR1、COX17)表达变化及细胞中ROS水平;应用real-time PCR检测铅暴露对大鼠脉络丛atp7a、ctr1、cox17的mRNA表达变化的影响;应用试剂盒检测铅暴露对大鼠皮质氧化损伤的影响;应用透射电子显微镜观察铅暴露对大鼠脉络丛上皮细胞超微结构的损伤。结果1铅暴露24周后,与对照组比较,300ppm、600ppm铅暴露组大鼠总运动距离、总穿格数、中央区域运动距离以及中央区域停留时间均降低(P0.05);铅暴露组大鼠第3、4天的逃避潜伏期均延长(P0.05),且600ppm铅暴露组第3、4天逃避潜伏期最长(P0.05);铅暴露组大鼠穿越平台次数均下降。尤其是600ppm铅暴露组大鼠穿台次数相比对照组减少了2.93次(P0.05)。2侧脑室灌注实验结果显示:脑脊液与灌注液的Cu64比值在20min达到平台期,达到平台期后铅暴露组大鼠收集液中Cu64比值为0.769±0.026,高于对照组的0.687±0.018(P0.05);铅暴露组大鼠BCB铜清除率低于对照组(P0.05);未见铅暴露组和对照组脑脊液与灌注液的C14比值有差异(P0.05)。3与对照组比较,铅暴露组大鼠全血、海马、皮质、脉络丛、脑脊液中铅、铜含量增加(P0.05);600ppm铅暴露组大鼠全血、海马、皮质、脉络丛、脑脊液中铅含量以及全血、海马、皮质的铜含量高于300ppm铅暴露组(P0.05)。4与对照组比较,铅暴露组大鼠皮质中AGEs含量增加,抑制羟自由基能力下降,GSH-ST活性下降,GSH含量降低(P0.05)。未见铅暴露后皮质中H2O2含量发生改变。5与对照组比较,铅暴露组大鼠脉络丛上皮细胞线粒体出现肿胀现象,部分线粒体Qg脊熔断,细胞间紧密连接缺失,部分细胞核发生变形。6铅暴露后,脉络丛上皮细胞中CTR1、ATP7A、COX17的荧光强度均高于对照组。7铅暴露组大鼠脉络丛上皮细胞中ROS水平与对照组比较,明显增加。8与对照组比较,铅暴露组大鼠脉络丛中ctr1、atp7a、cox17 mRNA表达水平均增加(P0.05)。结论铅暴露导致中枢神经系统铜稳态失调,氧化损伤增加,这可能与铅暴露后脉络丛上皮细胞超微结构损伤,铜转运蛋白和伴侣蛋白表达改变,进而导致血脑脊液屏障对铜清除能力下降有关。
[Abstract]:Objective to investigate the changes in copper content in the hippocampus, cortex, choroid plexus, blood and cerebrospinal fluid in the central nervous system of lead exposed rats, and the effect of lead exposure on the copper removal ability of the blood cerebrospinal fluid barrier (CSF), and the preliminary study of lead from the angle of CTR1 (ATP7A) and chaperone (COX17). The mechanism of copper accumulation in the central nervous system. The results of this study will further enrich the mechanism of lead neurotoxicity, and provide important information and targets for the study of biomarkers of lead neurotoxicity. Methods 45 healthy male SD rats were randomly divided into control group, 300ppm lead exposure group and 600ppm lead exposure group. After 7 days of adaptation, the results were passed. The free drinking water was given to lead exposed rats 300ppm, 600ppm and lead acetate drinking water. The control group was given 600ppm sodium acetate drinking water for 24 weeks. The rats' mental state, activity, diet and drinking water were observed daily, and the weight changes of rats were measured every week. After the end of 24 weeks, the Morris water fans were applied. The effects of lead and copper content in the whole blood, cortex, hippocampus, choroid plexus and cerebrospinal fluid were detected by ICP-MS, and the effects of lead exposure on the copper scavenging ability of the blood cerebrospinal fluid barrier in rats were detected by the lateral ventricle perfusion method, and the copper related eggs of choroid plexus epithelial cells were observed by laser scanning confocal microscope. The expression of white (ATP7A, CTR1, COX17) expression and the level of ROS in cells; the effect of real-time PCR on the expression of ATP7A, Ctr1 and COX17 in the choroid plexus of rats by real-time PCR; the effect of lead exposure on the oxidative damage in the cortex of rats was detected by the application of the kit; the ultrastructural observation of lead exposure on the ultrastructure of the rat choroid plexus epithelial cells was observed by transmission electron microscopy Results after 1 lead exposure for 24 weeks, compared with the control group, the total movement distance, total puncture number, central regional movement distance and central area residence time in the 300ppm and 600ppm lead exposed rats were all decreased (P0.05); the escape latency of the lead exposed rats on 3,4 days was prolonged (P0.05), and the 600ppm lead exposed group 3,4 days escape latency. The longest (P0.05) in the lead exposure group decreased the number of cross platform, especially in the 600ppm lead exposure group, the number of rats in the exposure group decreased by 2.93 times compared with the control group (P0.05).2 lateral ventricle perfusion test, which showed that the Cu64 ratio of cerebrospinal fluid and perfusion solution at 20min reached the platform stage, and the Cu64 ratio in the rat collection solution after the platform stage was 0. .769 + 0.026, higher than the control group 0.687 + 0.018 (P0.05), lead exposure group BCB copper clearance rate was lower than the control group (P0.05), no lead exposure group and the control group of cerebrospinal fluid and perfusion C14 ratio is different (P0.05).3 compared with the control group, lead exposed group of rats whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid lead, copper content increase (P0.05); 600pp (P0.05); 600pp (P0.05); 600pp The lead content in the whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid and the content of copper in the whole blood, hippocampus, and cortex of M lead exposed rats were higher than that of 300ppm lead exposure group (P0.05).4 and the control group. The content of AGEs in the cortex of lead exposed rats increased, the ability to inhibit the hydroxyl radical decreased, the GSH-ST activity decreased, and the GSH content decreased (P0.05). No lead exposed skin was found. Compared with the control group, the content of H2O2 in the cytoplasm was compared with the control group. The mitochondria of the choroid plexus epithelial cells in the lead exposed rats were swollen, some of the mitochondrial Qg ridges were broken, the close connections between the cells were missing, and the.6 lead exposure in some nuclei of the nuclei of the nuclei, and the fluorescence intensity of CTR1, ATP7A and COX17 in the choroid plexus epithelial cells was higher than that of the control group of.7 lead exposure. Compared with the control group, the level of ROS in the choroid plexus epithelial cells of the rats was significantly increased by.8. The expression level of Ctr1, ATP7A and COX17 mRNA in the choroid plexus of the lead exposed rats increased (P0.05). Conclusion lead exposure leads to the imbalance of copper in the central nervous system and the increase of oxidative damage, which may be associated with the ultrastructure of the choroid plexus epithelial cells after lead exposure. Structural damage, altered expression of copper transporters and chaperone proteins, leading to a decrease in blood cerebrospinal fluid barrier to copper clearance.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
本文编号：2144056
[Abstract]:Objective to investigate the changes in copper content in the hippocampus, cortex, choroid plexus, blood and cerebrospinal fluid in the central nervous system of lead exposed rats, and the effect of lead exposure on the copper removal ability of the blood cerebrospinal fluid barrier (CSF), and the preliminary study of lead from the angle of CTR1 (ATP7A) and chaperone (COX17). The mechanism of copper accumulation in the central nervous system. The results of this study will further enrich the mechanism of lead neurotoxicity, and provide important information and targets for the study of biomarkers of lead neurotoxicity. Methods 45 healthy male SD rats were randomly divided into control group, 300ppm lead exposure group and 600ppm lead exposure group. After 7 days of adaptation, the results were passed. The free drinking water was given to lead exposed rats 300ppm, 600ppm and lead acetate drinking water. The control group was given 600ppm sodium acetate drinking water for 24 weeks. The rats' mental state, activity, diet and drinking water were observed daily, and the weight changes of rats were measured every week. After the end of 24 weeks, the Morris water fans were applied. The effects of lead and copper content in the whole blood, cortex, hippocampus, choroid plexus and cerebrospinal fluid were detected by ICP-MS, and the effects of lead exposure on the copper scavenging ability of the blood cerebrospinal fluid barrier in rats were detected by the lateral ventricle perfusion method, and the copper related eggs of choroid plexus epithelial cells were observed by laser scanning confocal microscope. The expression of white (ATP7A, CTR1, COX17) expression and the level of ROS in cells; the effect of real-time PCR on the expression of ATP7A, Ctr1 and COX17 in the choroid plexus of rats by real-time PCR; the effect of lead exposure on the oxidative damage in the cortex of rats was detected by the application of the kit; the ultrastructural observation of lead exposure on the ultrastructure of the rat choroid plexus epithelial cells was observed by transmission electron microscopy Results after 1 lead exposure for 24 weeks, compared with the control group, the total movement distance, total puncture number, central regional movement distance and central area residence time in the 300ppm and 600ppm lead exposed rats were all decreased (P0.05); the escape latency of the lead exposed rats on 3,4 days was prolonged (P0.05), and the 600ppm lead exposed group 3,4 days escape latency. The longest (P0.05) in the lead exposure group decreased the number of cross platform, especially in the 600ppm lead exposure group, the number of rats in the exposure group decreased by 2.93 times compared with the control group (P0.05).2 lateral ventricle perfusion test, which showed that the Cu64 ratio of cerebrospinal fluid and perfusion solution at 20min reached the platform stage, and the Cu64 ratio in the rat collection solution after the platform stage was 0. .769 + 0.026, higher than the control group 0.687 + 0.018 (P0.05), lead exposure group BCB copper clearance rate was lower than the control group (P0.05), no lead exposure group and the control group of cerebrospinal fluid and perfusion C14 ratio is different (P0.05).3 compared with the control group, lead exposed group of rats whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid lead, copper content increase (P0.05); 600pp (P0.05); 600pp (P0.05); 600pp The lead content in the whole blood, hippocampus, cortex, choroid plexus, cerebrospinal fluid and the content of copper in the whole blood, hippocampus, and cortex of M lead exposed rats were higher than that of 300ppm lead exposure group (P0.05).4 and the control group. The content of AGEs in the cortex of lead exposed rats increased, the ability to inhibit the hydroxyl radical decreased, the GSH-ST activity decreased, and the GSH content decreased (P0.05). No lead exposed skin was found. Compared with the control group, the content of H2O2 in the cytoplasm was compared with the control group. The mitochondria of the choroid plexus epithelial cells in the lead exposed rats were swollen, some of the mitochondrial Qg ridges were broken, the close connections between the cells were missing, and the.6 lead exposure in some nuclei of the nuclei of the nuclei, and the fluorescence intensity of CTR1, ATP7A and COX17 in the choroid plexus epithelial cells was higher than that of the control group of.7 lead exposure. Compared with the control group, the level of ROS in the choroid plexus epithelial cells of the rats was significantly increased by.8. The expression level of Ctr1, ATP7A and COX17 mRNA in the choroid plexus of the lead exposed rats increased (P0.05). Conclusion lead exposure leads to the imbalance of copper in the central nervous system and the increase of oxidative damage, which may be associated with the ultrastructure of the choroid plexus epithelial cells after lead exposure. Structural damage, altered expression of copper transporters and chaperone proteins, leading to a decrease in blood cerebrospinal fluid barrier to copper clearance.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114
【参考文献】
相关期刊论文 前10条
1 周婷;王婉宜;孙晓蒙;刘元元;;中国7～18岁儿童青少年超重肥胖危险因素meta分析[J];中国公共卫生;2016年10期
2 苏红;高晶;尹镜雯;刘楠;曹福源;薛玲;高福佳;李清钊;张艳淑;姚林;;纳米铅和常规铅暴露对仔鼠海马及皮质氧化损伤的比较研究[J];环境与健康杂志;2015年01期
3 张莹;焦怡琳;陆凯;何广学;;中国成年人超重肥胖影响因素meta分析[J];中国公共卫生;2015年02期
4 刘洪娟;吕翠;刘晓丽;张文生;;铅毒性拮抗剂的研究进展[J];环境与职业医学;2014年11期
5 王健辉;程肖蕊;周文霞;张永祥;;β分泌酶生物学特性的研究进展[J];国际药学研究杂志;2014年04期
6 杨汉策;王永伟;杨跃林;兰亚佳;唐成志;;累积铅暴露工人ALAD基因多态性与肾损害的相关分析[J];预防医学情报杂志;2014年05期
7 杜欢;郭茂娟;姜希娟;王一婧;胡先同;;RAGE与阿尔茨海默病关系的研究进展[J];现代生物医学进展;2014年14期
8 张园;耿春女;蔡超;;铅暴露对人体健康风险评价的模型综述[J];环境化学;2013年06期
9 席永兵;袁淑娟;路小婷;;铅对肾细胞毒性作用的研究[J];中国药物与临床;2012年07期
10 李平;李芬;叶f ;吕玲;陈军;;Nrf 2信号通路在铅致SH-SY5Y细胞氧化应激中作用[J];中国公共卫生;2012年07期
相关会议论文 前1条
1 张艳淑;杨辉;闫立成;姚林;李清钊;郑伟;;铜稳态失调在铅暴露导致大鼠损伤中的作用[A];中华预防医学会自由基预防医学专业委员会2012年夏季学术交流会论文集[C];2012年
相关硕士学位论文 前1条
1 冯佩佩;醋酸铅与纳米硫化铅暴露对大鼠血脑脊液屏障损伤的比较研究[D];华北理工大学;2016年
，本文编号：2144056
本文链接：https://www.wllwen.com/yixuelunwen/yufangyixuelunwen/2144056.html
