酒精所致氧化应激对高尔基体SPCA1的影响及神经生长因子的保护作用

发布时间：2018-02-21 07:22

本文关键词： 酒精 SPCA1 高尔基体 Ca~(2+) NAC NGF　出处：《中南大学》2013年博士论文　论文类型：学位论文

【摘要】：目的：本论文研究旨在探索氧化应激是否介导酒精对高尔基体SPCAl的影响,以及神经生长因子是否具备改善酒精毒性的作用,为酒精相关疾病治疗提供新思路。方法： 1.培养小鼠神经细胞瘤N2A细胞,建立酒精封闭系统； 2.实验分三个模块：急性酒精作用模块、抗氧化剂NAC预处理模块、神经生长因子NGF预处理模块； 3.光学显微镜、MTT法评价急性酒精作用对N2A细胞生存的影响； 4.荧光显微镜、流式细胞仪观察及检测细胞内氧化应激水平； 5.多功能酶标仪检测细胞内游离钙离子浓度的变化； 6.实时荧光定量PCR检测基因ATP2C1表达变化； 7. Western blot检测蛋白SPCAl表达变化。结果： 1.急性酒精作用后,小鼠N2A细胞形态结构无明显变化,MTT吸光度值变化无统计学差异,说明酒精200mM浓度以下24h作用对小鼠神经细胞瘤N2A细胞生存无明显影响； 2.急性酒精作用后,各浓度酒精亚组均较空白组细胞荧光强度高,且随酒精浓度上升逐渐增大,在酒精100mM浓度荧光强度增幅最大(16.3%),随后小幅下降。NAC预处理后,NAC1mM、2mM浓度下细胞内氧化应激相比单独酒精作用无明显变化,仅在NAC4mM浓度下细胞内氧化应激较单独酒精作用组低,降幅为3.7%。 3.急性酒精作用后,各浓度酒精亚组均较空白组细胞内游离钙离子浓度增高,且随酒精浓度上升逐渐增大,在100mM浓度时增幅最大(22.4%),随后小幅下降。NAC预处理后,各浓度NAC亚组细胞内游离钙离子浓度均较单独酒精作用组明显降低,且下降程度随NAC浓度上升逐渐增大,在4mM浓度降幅最大(31.8%)。NGF预处理后,各浓度NGF亚组的细胞内游离钙离子浓度均较单独酒精作用组明显降低,且下降程度随NGF浓度上升逐渐增大,在50ng/ml浓度时降幅最大(31.4%),随后小幅下降。 4.急性酒精作用后,各浓度酒精亚组基因ATP2C1、蛋白SPCA1表达水平均较空白组升高,且随酒精浓度上升逐渐增大,在200mM浓度时达最大增幅(45%,34.9%)。NAC预处理后,各浓度NAC亚组基因ATP2C1、蛋白SPCA1表达水平均较单独酒精作用亚组下降,且下降程度随NAC浓度升高而增大,在4mM浓度下降幅度最大(30%,12%)。NGF预处理后,各浓度NGF亚组基因ATP2C1、蛋白SPCA1表达水平均较单独酒精作用亚组升高,且上升程度随NGF浓度升高而增大,在100ng/ml浓度时表达最高(16%,6%)。结论： 1.急性酒精作用导致了细胞内氧化应激、钙超载,上调了SPCA1的表达。 2.NAC预处理可缓解急性酒精所致细胞内氧化应激、钙超载,下调SPCAl表达。 3.NGF预处理可上调SPCAl表达,缓解急性酒精所致细胞内钙超载。图17幅,表12张,参考文献105篇
[Abstract]:Objective:. The purpose of this study is to explore whether oxidative stress mediates the effect of alcohol on Golgi body SPCAl and whether nerve growth factor can improve alcohol toxicity and provide new ideas for the treatment of alcohol-related diseases. Methods:. 1. N2A cells were cultured and the alcohol blocking system was established. 2. The experiment was divided into three modules: acute alcohol action module, antioxidant NAC pretreatment module, nerve growth factor NGF pretreatment module; 3. MTT assay was used to evaluate the effect of acute alcohol on the survival of N2A cells. 4. Fluorescence microscope and flow cytometry were used to observe and detect the level of intracellular oxidative stress. 5. The change of intracellular free calcium ion concentration was detected by multifunctional enzyme labeling instrument. 6. Real-time quantitative PCR was used to detect the change of gene ATP2C1 expression. 7. The expression of protein SPCAl was detected by Western blot. Results:. 1. After acute alcohol treatment, there was no significant change in morphology and structure of N2A cells. There was no significant difference in MTT absorbance value, indicating that the survival of N2A cells was not significantly affected by the concentration of 200 mm alcohol for 24 hours. 2.After the acute alcohol treatment, the fluorescence intensity of the cells in each concentration of alcohol subgroup was higher than that in the blank group, and gradually increased with the increase of alcohol concentration. The maximum increase of fluorescence intensity was 16.3mm in 100 mm alcohol concentration, and then decreased slightly. After pretreatment with NAC, the oxidative stress of NAC _ 1mMN _ 2 mm concentration had no significant change compared with that of alcohol alone, but the intracellular oxidative stress was lower at the concentration of NAC4mM than that in the group treated with alcohol alone. The decline was 3.7. 3. After acute alcohol treatment, the concentration of intracellular free calcium in each concentration of alcohol subgroup was higher than that in the blank group, and gradually increased with the increase of alcohol concentration, the largest increase was 22.4% at 100 mm concentration, and then decreased slightly after pretreatment with NAC. The intracellular free calcium concentration in each concentration of NAC subgroup was significantly lower than that in alcohol alone group, and the degree of decrease increased with the increase of NAC concentration. The intracellular free Ca ~ (2 +) concentration in each NGF subgroup was significantly lower than that in the alcohol alone group, and the degree of decrease increased with the increase of NGF concentration, the largest decrease was 31.4% at the concentration of 50 ng / ml, and then decreased slightly. 4. After acute alcohol treatment, the expression level of ATP2C1 and protein SPCA1 in each concentration of alcohol subgroup was higher than that in the control group, and gradually increased with the increase of alcohol concentration, and the maximum increase was reached at 200mm concentration after pretreatment with ATP2C1 and NAC. The expression level of ATP2C1 and protein SPCA1 in each concentration of NAC subgroup was lower than that in the single alcohol subgroup, and the degree of decrease increased with the increase of NAC concentration. After pretreatment with 4mm concentration of ATP2C1, ATP2C1 and protein, the concentration of ATP2C1 and protein increased with the increase of NAC concentration. The expression level of ATP2C1 and protein SPCA1 in each NGF subgroup was higher than that in alcohol group alone, and the degree of increase was increased with the increase of NGF concentration. The highest expression of ATP2C1 and protein SPCA1 was observed at 100ng / ml. Conclusion:. 1. Acute alcohol induced intracellular oxidative stress, calcium overload and up-regulation of SPCA1 expression. 2. NAC pretreatment alleviated intracellular oxidative stress induced by acute alcohol, calcium overload and down-regulated SPCAl expression. 3. NGF pretreatment can up-regulate the expression of SPCAl and relieve intracellular calcium overload induced by acute alcohol.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R749.62

【参考文献】