元素组在铅暴露致神经损伤中的作用研究

发布时间：2019-06-02 16:17

【摘要】：目的本研究主要探讨铅暴露后海马、皮质和脑脊液变化的元素图谱,同时初步阐明铜和铜转运蛋白在铅神经毒性中铜蓄积的作用,以期为铅中毒的防治提供理论依据。方法1动物模型建立21天雄性SD大鼠80只随机分为铅暴露组和对照组,通过自由饮水方式染毒,铅暴露组饮用含有200mg/L醋酸铅。染毒时间分为3、6、9、12周。应用Morris水迷宫进行神经行为学测试。2细胞模型的建立大鼠神经胶质瘤细胞系C6,应用0或10μmol·L-1醋酸铅处理24h,然后更换含有2μmol·L-1的氯化铜培养基,培养0、0.5、1、2、4、8h后收集细胞,CCK-8法检测细胞存活率。3采用电感耦合等离子体质谱法检测铅暴露后大鼠海马、皮质、脑脊液中15种元素的含量和C6细胞中铅、铜的含量。4荧光实时定量PCR检测铅暴露后细胞中CTR1、DMT1、CCS、ATOX1、COX17、ATP7A、ATP7B的mRNA表达。5激光共聚焦检测铅暴露后C6细胞中CTR1和ATP7A的表达。结果1实验大鼠的一般情况:在染毒期间,所有实验大鼠摄食和饮水量未见异常。饲养过程中,实验大鼠体重正常增长,各组体重增加变化无差异。2铅暴露第6周到第12周,大鼠的逃逸潜伏期与对照组比较显著增长(P0.05)。3铅暴露后大鼠海马、皮质、脑脊液中主要差异元素组:铅暴露后大鼠皮质中主要差异元素包括Ca、Fe、Cu;海马中主要的差异元素包括Na、Mg、Al、Cu、Zn;脑脊液中主要差异元素包括Na、Al、Cu、Fe、V、Ni。铜在暴露大鼠海马、皮质、脑脊液中均有显著变化,是重要的差异元素。4铅暴露导致C6细胞内铜蓄积:C6细胞铅暴露24h后,细胞内铅和铜的浓度分别为0.19±0.01(μg/mg pro)和0.51±0.05(μg/mg pro),是对照组的1.98和1.23倍,差异有统计学意义(P0.05)。C6细胞在含铜培养基培养0.5,1,2,4,8h后,铜含量都随着时间的延长而上升,在0.5h,1h,2h和8h时,细胞中铜的浓度分别是对照组的1.12、1.35、1.19、1.13倍(P0.05);其中,在培养1h后,铅暴露组细胞铜吸收速率最快(P0.05)。5铜转运相关蛋白在铅暴露所致铜蓄积中的作用:C6细胞在铅暴露24h后,吸收铜0h时,CTR1和DMT1的mRNA表达比对照组分别增加了12%和36%,ATP7AmRNA的表达降低了25%,差异具有统计学意义(P0.05);同时,激光共聚焦结果显示铅暴露24h后,C6细胞中CTR1表达的荧光强度高于对照组,ATP7A的荧光强度低于对照组。6铅暴露后继续吸收铜8h时,C6细胞内ATP7AmRNA表达明显升高,COX17mRNA表达明显降低,差异有统计学意义。结论1铅暴露可导致海马、皮质和脑脊液中差异图谱的变化不同,但是铜是主要的差异元素。2铅暴露后可导致C6细胞中铜蓄积,这与铜转入蛋白CTR1和DMT1的表达增加和铜转出蛋白ATP7A的表达下降有关。
[Abstract]:Objective to investigate the elemental profiles of hippocampal, cortical and cerebrospinal fluid changes after lead exposure, and to elucidate the role of copper and copper transporter in copper accumulation in lead neurotoxicity, so as to provide theoretical basis for the prevention and treatment of lead poisoning. Methods 1 80 male SD rats were randomly divided into lead exposure group and control group. Lead exposure group was exposed to free drinking water. Lead exposure group drank lead acetate containing 200mg/L. The exposure time was divided into 3, 6, 9, 12 weeks. The neurobehavioral test was carried out by Morris water maze. 2 the rat glioma cell line C6was established. The rat glioma cell line C6was treated with 0 or 10 渭 mol 路L-1 lead acetate for 24 h, and then the copper chloride medium containing 2 渭 mol 路L-1 was replaced and cultured for 0, 0.5, 1. The cell survival rate was measured by CCK-8 assay. 3 the contents of 15 elements in hippocampus, cortex and cerebrospinal fluid of rats exposed to lead and the content of lead in C6 cells were detected by inductively coupled plasma mass spectrometry (ICP-MS). The content of copper was detected by fluorescence real-time quantitative PCR. The mRNA expression of CTR1,DMT1,CCS,ATOX1,COX17,ATP7A,ATP7B in C6 cells exposed to lead was detected by fluorescence real-time quantitative PCR. 5 the expression of CTR1 and ATP7A in C6 cells exposed to lead was detected by confocal laser. Results 1 the general situation of experimental rats: during the exposure period, there was no abnormal intake and drinking amount of food and water in all experimental rats. During the feeding process, the body weight of the experimental rats increased normally, and there was no difference in the weight gain of each group. 2 at the 6th to 12th week of lead exposure, the escape latency of the rats was significantly higher than that of the control group (P 0.05). 3 after lead exposure, The main difference elements in the cortex and cerebrospinal fluid: the main difference elements in the cortex of rats after lead exposure include Ca,Fe,Cu; The main difference elements in hippocampus include Na,Mg,Al,Cu,Zn;, cerebrospinal fluid, Na,Al,Cu,Fe,V,Ni.. Copper has significant changes in hippocampus, cortex and cerebrospinal fluid of rats exposed to lead, which is an important differential element. 4 lead exposure leads to copper accumulation in C6 cells: 24 hours after exposure to lead in C6 cells, The intracellular concentrations of lead and copper were 0.19 卤0. 01 (渭 g/mg pro) and 0. 51 卤0. 05 (渭 g/mg pro), respectively, which were 1.98 and 1.23 times higher than those in the control group, respectively. The difference was statistically significant (P 0.05). C6 cells were cultured in copper medium 0.5, 1, 2, 4, 8 h, and the copper content increased with the prolongation of time, 0.5 h, 1 h, 2 h and 8 h, respectively. The concentration of copper in the cells was 1.12, 1.35, 1.19 and 1.13 times higher than that in the control group (P 0.05). Among them, after 1 hour of culture, the copper absorption rate of lead exposed group was the fastest (P 0.05). 5 the role of copper transport related proteins in copper accumulation induced by lead exposure: C 6 cells absorbed copper for 0 h after exposure to lead for 24 h. Compared with the control group, the mRNA expression of CTR1 and DMT1 increased by 12% and 36%, respectively, and the expression of ATPase AmRNA decreased by 25%, the difference was statistically significant (P 0.05). At the same time, the results of laser confocal showed that the fluorescence intensity of CTR1 expression in C6 cells was higher than that in the control group, and the fluorescence intensity of ATP7A was lower than that in the control group. 6 when copper was absorbed for 8 hours after lead exposure, the expression of ATP7AmRNA in C6 cells increased significantly. The expression of COX17mRNA was significantly decreased, the difference was statistically significant. Conclusion 1 lead exposure can lead to different changes of difference map in hippocampus, cortex and cerebrospinal fluid, but copper is the main difference element. 2 lead exposure can lead to copper accumulation in C6 cells. This is related to the increase of the expression of copper transfer protein CTR1 and DMT1 and the decrease of the expression of copper transfer protein ATP7A.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R114

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