氯化镧导致原代大鼠皮层神经元损伤作用的研究

发布时间：2018-02-03 02:34

本文关键词： 镧神经元活性氧线粒体 bcl-2 bax 细胞凋亡　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 前言稀土(rare earth, RE)包括17种元素,其中镧至铕为轻稀土元素,钆至钇为重稀土元素。我国是稀土资源最多的国家,近年来随着稀土在农业、畜牧业、工业、国防和高技术产业及现代生物医学上应用的日益扩展,稀土元素正在广泛进入环境,并通过食物链进入人体。因此,稀土对生态环境和人体健康的影响及其生物学效应,已成为不容忽视的卫生学问题。已有报道表明RE具有神经毒性。稀土矿区流行病学调查显示,长期摄入RE使儿童智商和记忆力下降,从而危及矿区儿童的基本素质,可能造成深远影响。动物实验也证明RE影响学习记忆,可能涉及降低抗氧化能力；干扰脑内微量元素的代谢；影响酶活力和神经递质的释放等。镧作为轻稀土元素的代表,化学性质比较活泼,动物实验表明镧具有神经毒性,但其作用机制尚不清楚。本研究以大鼠皮层神经元为研究对象,观察不同剂量镧处理组神经元的线粒体活性,细胞内活性氧含量,线粒体膜电位和bcl-2、baxmRNA表达的改变以及细胞凋亡的变化情况,来探讨氯化镧对神经元的凋亡及其线粒体功能的影响,为阐明稀土元素的神经毒性机制提供新的实验证据。实验分组 LaCl3用无血清DMEM配制,终浓度分别为0.01,0.1,1.0,2.0mmol/L,对照组应用无血清DMEM。研究方法 1、神经元免疫细胞化学法纯度鉴定 2、MTT法测定神经元线粒体活性 3、DCFH-DA荧光探针法测定细胞内ROS含量 4、Rhodamine123荧光探针法测定神经元线粒体膜电位 5、RT-PCR法测定神经元bcl-2, bax mRNA表达 6、流式细胞仪法测定细胞凋亡率 7、倒置显微镜观察神经元形态学改变结果 1、神经元的鉴定显微镜下可见绝大部分细胞胞浆被染成棕黄色,每张玻片选择10个视野,计数阳性细胞比例,免疫阳性细胞可达90%以上,因此可以将此原代培养细胞作为神经元的体外模型。 2、神经元线粒体活性的改变镧暴露后细胞线粒体活性下降,各剂量组与对照组相比差异均显著(p0.01),且呈现一定剂量—反应关系。 3、细胞内活性氧含量的改变镧暴露后细胞内ROS水平呈升高趋势,各剂量组细胞内ROS水平均高于对照组且统计学差异显著(p0.05)。 4、线粒体膜电位变化镧暴露组细胞线粒体膜电位均降低,并随镧暴露浓度的增加而降低显著,与对照组比较有明显差异(p0.05)。 5、bcl-2、bax mRNA表达改变镧暴露组神经元bax mRNA表达增多,同时bcl-2 mRNA表达减少,0.1和1.0mmol/L组与对照组比较均存在显著差异(p0.01)。 6、细胞凋亡流式检测氯化镧暴露组细胞凋亡率呈升高趋势,且0.1和1.0mmol/L剂量组与对照组差异显著(p0.01)；细胞坏死率也有增加,但差异不明显,且低于凋亡率。结论 1、氯化镧能够使神经元内ROS水平升高,神经元线粒体活性下降,线粒体跨膜电位降低,损伤线粒体功能。 2、氯化镧可使凋亡调控基因bax mRNA表达增多,而bcl-2 mRNA表达减少,并可诱导大鼠皮层神经元凋亡。
[Abstract]:Preface
Rare earth (rare earth, RE) includes 17 elements, including lanthanum to EU for light rare earth elements, yttrium gadolinium to heavy rare earth elements. As our country is the most rare earth resources in recent years, with the rare earth elements in agriculture, animal husbandry, industry, expanding application of national defense and high technology industry and modern medicine. Rare earth elements are widely into the environment, and enter the body through the food chain. Therefore, effect of Rare Earths on the ecological environment and human health and biological effects, has become a health problem can not be ignored.
It has been reported that RE have neurotoxicity. Epidemiological survey of mining area of rare earth, long-term intake of RE children IQ and memory decline, thereby endangering children's basic quality of mining area, may have an impact. Animal experiments also show that RE on learning and memory, may involve lower antioxidant capacity; trace element interference in brain metabolism; enzyme activity and influence the release of neurotransmitters.
La as a representative of light rare earth elements, the chemical nature is lively, the animal experiment showed that lanthanum has neurotoxicity, but its mechanism is still unclear. In this study, rat cortical neurons as the research object, the effects of different doses of La group neuron mitochondrial activity, active oxygen content in cells, mitochondrial membrane potential and Bcl-2 changes the change of baxmRNA expression and cell apoptosis, to investigate the effect of lanthanum chloride on neuronal apoptosis and mitochondrial function, to provide new experimental evidence for clarifying the mechanism of neurotoxicity of rare earth elements.
Experimental grouping
LaCl3 was prepared with serum-free DMEM, the final concentration was 0.01,0.1,1.0,2.0mmol/L, and the control group was serum-free DMEM.
research method
1, the purity identification of neuron immunocytochemical method
2, MTT assay for the determination of neuronal mitochondrial activity
Determination of ROS content in cells by 3, DCFH-DA fluorescence probe
4, Rhodamine123 fluorescence probe method for the determination of mitochondrial membrane potential in neurons
5, RT-PCR method was used to determine the expression of Bcl-2 and Bax mRNA in neurons
6, determination of apoptosis rate by flow cytometry
7, the morphological changes of neurons were observed by inverted microscope
Result
1, identification of neurons
Under microscope, most of the cytoplasm was dyed brown. Each slide selected 10 fields of view, counting the proportion of positive cells, and immunoreactive cells could reach more than 90%. Therefore, this primary culture cell can be used as an in vitro model of neurons.
2, changes in the activity of mitochondria in neurons
After lanthanum exposure, the cell mitochondrial activity decreased, and the difference in each dose group was significantly higher than that of the control group (P0.01), and there was a certain dose response relationship.
3, changes in the content of active oxygen in cells
After lanthanum exposure, the intracellular ROS level increased, and the intracellular ROS level in each dose group was higher than that in the control group and the statistical difference was significant (P0.05).
4, the change of mitochondrial membrane potential
The cell mitochondrial membrane potential in the Lanthanum Exposure group decreased and decreased significantly with the increase of lanthanum exposure concentration, which was significantly different from the control group (P0.05).
Changes in expression of 5, Bcl-2, Bax mRNA
The expression of Bax mRNA in the lanthanum exposed group increased and the expression of Bcl-2 mRNA decreased. There was a significant difference between the 0.1 and 1.0mmol/L groups (P0.01).
6, flow cytometry of cell apoptosis
The apoptosis rate of lanthanum chloride exposure group showed an increasing trend, and there was a significant difference between the 0.1 and 1.0mmol/L dose group and the control group (P0.01), and the cell necrosis rate also increased, but the difference was not obvious, and it was lower than the apoptosis rate.
conclusion
1, lanthanum chloride can increase the level of ROS in neurons, decrease the activity of mitochondria in neurons, decrease the mitochondrial transmembrane potential and damage the function of mitochondria.
2, lanthanum chloride can increase the expression of apoptosis regulating gene Bax mRNA, and decrease the expression of Bcl-2 mRNA, and induce apoptosis of rat cortical neurons.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R363

【参考文献】