亚慢性砷暴露对小鼠脑组织RXR表达的影响

发布时间：2018-06-01 06:32

本文选题：三氧化二砷 + 神经毒性　；参考：《大连医科大学》2012年硕士论文

【摘要】：研究背景：砷（Arsenic, As）是一种广泛分布于土壤、岩石和水环境中的有毒类金属元素。As在自然界中主要以化合物的形式存在，对健康具有多方面的危害，可引发多器官和多系统的形态学和功能上的异常改变。As对神经系统的影响主要表现为头痛、嗜睡、烦躁、记忆力减退、定向力障碍，惊厥甚至昏迷等亚临床的神经损伤。儿童由于正处于生理发育阶段对外源化合物的毒作用较为敏感。大量流行病学调查、动物实验表明，As暴露儿童的中枢神经系统损伤主要表现为学习记忆能力的损害，已广泛引起社会的高度关注，但目前，关于砷致学习记忆能力损害的分子作用机制尚不清楚。本课题组的前期研究发现，砷暴露可显著抑制脑组织中钙离子/钙调蛋白依赖性蛋白激酶Ⅳ(calcium/calmodulin-dependent protein kinaseⅣ, Camk4)基因、蛋白的表达及c-Fos、JunB等与学习记忆相关基因、蛋白的表达，破坏脑组织中长时程记忆（LTM）的形成。据文献报道，脑组织Camk4基因的转录和翻译依赖于两种核受体即甲状腺激素受体（TR）和视黄醇X受体(RXR)的存在。我们的前期研究已经发现，砷暴露小鼠大、小脑组织TR受体亚型之一TRβ在基因、蛋白水平均表达显著下调，，但砷是否也影响RXR基因表达国内外未见相关报道。目的：观察砷对小鼠大、小脑组织中RXR基因和蛋白的表达影响，为阐明砷的神经毒作用机制以及防治砷的神经毒性危害提供靶基因依据。方法：SPF级小鼠40只，按体重将小鼠随机分为饮用水对照组、1ppm As_2O_3染毒组、2ppm As_2O_3染毒组、4ppm As_2O_3染毒组、4ppm As_2O_3+150mg/kg牛磺酸保护组。通过自然饮用含不同浓度As_2O_3蒸馏水的方式使小鼠染砷，保护剂是以灌胃方式给予，连续染毒60天后取脑组织。用基因芯片技术和PCR技术检测小鼠脑组织视黄醇X受体(RXR)的差异表达，并进一步用Western blot和免疫组化的方法证实RXR蛋白水平表达和组织分布的改变。采用SPSS11.5统计软件，用单因素方差分析（ANOVA），比较各染砷组与对照组间的统计学差异，两组间比较用LSD法分析，以P0.05表示统计学差异显著。结果：基因芯片结果显示，与对照组比较，各砷暴露组大脑组织RXR基因表达均显著上调，尤其4ppm组上调更明显(P 0.05)，而各组之间，小鼠小脑组织RXR的基因表达差异无统计学意义(P0.05)。Real Time PCR定量检测结果显示，砷暴露组大脑中RXR基因表达显著上调，差异有统计学意义(P 0.05)，与基因芯片结果一致。同时，Western blotting检测结果显示，砷暴露导致小鼠大脑RXR蛋白表达上调，差异有统计学意义(P 0.05)，且呈剂量-反应关系。免疫组化显示RXR在砷染毒小鼠大脑4ppm组高表达。另外，我们发现，在砷暴露小鼠大脑组织，抗氧化剂牛磺酸对砷致2ppm和4ppm剂量组的RXR基因和蛋白表达上调有拮抗作用(P 0.05)。结论：在亚慢性砷暴露可上调小鼠大脑组织中RXR基因表达水平，但在小脑组织中，RXR基因表达水平并无显著差异变化。牛磺酸对亚慢性砷暴露使小鼠大脑组织中RXR基因和蛋白的表达上调有拮抗作用。提示，亚慢性砷暴露导致脑组织中RXR基因异常表达可能与砷诱导的氧化应激作用有关。
[Abstract]:Research background: Arsenic (As) is a kind of toxic metal element, which is widely distributed in soil, rock and water environment,.As exists mainly in the form of compound in nature, and has many harmful effects on health. It can cause the abnormal changes of morphology and energy of multiple organs and multiple systems and the influence of.As on the nervous system. It is the subclinical nerve injury of headache, drowsiness, irritability, memory loss, disorder of orienteering, convulsion and even coma. Children are more sensitive to the toxic effects of exogenous compounds at the stage of physiological development. A large number of epidemiological investigations have shown that the central nervous system injury in children exposed to As is mainly learning and memory. The damage of capacity has aroused great concern in society, but at present, the molecular mechanism of arsenic induced impairment of learning and memory is not clear. The previous study in our group found that arsenic exposure could significantly inhibit the calcium ion / calmodulin dependent protein kinase IV (calcium/calmodulin-dependent protein kinase IV, C) in brain tissue. Amk4) gene, expression of protein and c-Fos, JunB, and learning and memory related genes, protein expression, destroying the formation of long history memory (LTM) in the brain tissue. It is reported that the transcription and translation of the Camk4 gene of the brain is dependent on the existence of two nuclear receptors, the thyroid hormone receptor (TR) and the retinol X receptor (RXR). Our preliminary study has already been reported. It was found that arsenic exposure in mice was large, and TR beta, one of the TR receptor subtypes of cerebellar tissue, decreased significantly in gene and protein levels, but whether arsenic also affects the expression of RXR gene has not been reported at home and abroad.
Objective: To observe the effect of arsenic on the expression of RXR gene and protein in the large and cerebellar tissues of mice, and to provide the target gene for elucidating the mechanism of arsenic neurotoxicity and the prevention and treatment of arsenic neurotoxicity.
Methods: 40 mice of grade SPF were randomly divided into drinking water control group, 1ppm As_2O_3 dye group, 2ppm As_2O_3 dye group, 4ppm As_2O_3 poisoning group, 4ppm As_2O_3+150mg/kg taurine protection group. The mice were exposed to arsenic by natural drinking with different concentrations of As_2O_3 distilled water, and the protective agent was given by gavage and continuous dyeing. The brain tissue was extracted after 60 days. The differential expression of retinol X receptor (RXR) in mouse brain tissue was detected by gene chip technology and PCR technique. The expression of RXR protein and the change of tissue distribution were confirmed by Western blot and immunohistochemistry. The SPSS11.5 statistics software was used to compare the arsenic staining groups with the single factor variance analysis (ANOVA). The statistical difference between the two groups was analyzed by LSD. P0.05 showed significant difference.
Results: the results of gene chip showed that the expression of RXR gene in the brain tissue of each arsenic exposure group was significantly up-regulated compared with the control group, especially in the 4ppm group (P 0.05), but there was no significant difference in the gene expression of RXR in the cerebellar tissues of the mice (P0.05).Real Time PCR quantitative detection results showed that the RXR base in the arsenic exposed group was in the brain. The difference was statistically significant (P 0.05), which was consistent with the results of gene chip. At the same time, the results of Western blotting detection showed that arsenic exposure led to the up regulation of RXR protein expression in the brain of mice (P 0.05), and showed a dose response relationship. Immunohistochemistry showed that RXR was highly expressed in the 4ppm group of arsenic exposed mice. In addition, we found that the antioxidant taurine had an antagonistic effect on the up regulation of RXR gene and protein expression in arsenic induced 2ppm and 4ppm doses in arsenic exposed mice brain tissue (P 0.05).
Conclusion: subchronic arsenic exposure can increase the level of RXR gene expression in the brain tissue of mice, but there is no significant difference in the expression of RXR gene in the cerebellar tissues. Taurine has antagonistic effect on the up regulation of RXR gene and protein expression in the brain tissue of mice. Abnormal expression of genes may be related to arsenic induced oxidative stress.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】