【作者】 陈洋；

【导师】 胡治平；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【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篇更多还原

【Abstract】 Objective:The objectives of this study were to clarify whether oxidative stress induced by ethanol destroys intracellular Ca2+homeostasis resulting in golgi apparatus SPCA1functional changes and whether NGF had a protective effect against ethanol.Methods1. We employed closed chamber system to mimic ethanol admiration in vitro.2. Experimental grouping was designed as follows:acute ethanol group, acute ethanol(100mM)+NAC group, acute ethanol(100mM)+NGF group.3. The neuronal viability was measured by the MTT assay, as well as the morphologic changes were studied under Light Microscopes.4. The oxidative stress was measured by flow cytometric analysis and fluorescence Microscope.5. The intracellular free Ca2+concentration was measured by multi-functional enzyme-labeling instrument6. The mRNA levels of SPCA1were tested by quantitative Real-Time polymerase chain reaction.7. The protein level of SPCA1was determined by western blotting.Result:1. After acute ethanol intake, there weren’t obvious cell morphology changes in ethanol subsets, and the optical density changes of ethanol subsets had non-statistical meaning comparing to the blank, which indicated acute ethanol intake had no effect on the viability of N2A cell.2. After acute ethanol intake, the fluorescence intensity of ethanol subsets were stronger than the blank, which were dependent of ethanol concentration with the strongest intensity at Ethanol100mM subset. In Ethanol(100mM)+NAC, the fluorescence intensity was only weaker at NAC4mM subset comparing to the ethanol intake subset.3. After acute ethanol intake, the intracellular free Ca2+concentration of ethanol subsets increased comparing to the blank, which were dependent of ethanol concentration with the highest concentration at Ethanol100mM subset. In Ethanol(100mM)+NAC, the intracellular free Ca2+concentrations decreased comparing to the ethanol intake, which were dependent of NAC concentration with the lowest concentration at NAC4mM subset. In Ethanol(100mM)+NGF, the intracellular free Ca2+concentration of NGF subsets decreased comparing to the ethanol intake subset, which were dependent of NGF concentration with the lowest concentration at NGF50ng/ml subset.4. After acute ethanol intake, the mRNA and protein levels of SPCA1of ethanol subsets increased comparing to the blank, which were dependent of ethanol concentration with the highest expression at Ethanol200mM subset. In Ethanol(100mM)+NAC, the mRNA and protein levels of SPCA1decreased comparing to the ethanol intake, which were dependent of NAC concentration with the lowest expression at NAC4mM subset. In Ethanol(100mM)+NGF, the mRNA and protein levels of SPCA1of NGF subsets increased comparing to the ethanol intake subset, which were dependent of NGF concentration with the highest expression at NGF100ng/ml subset.Conclusion:1. Acute ethanol intake induced oxidative stress, calcium overload and elevated the expression of SPCA1at mRNA and protein level in N2A cell.2. NAC pretreatment alleviated oxidative stress and calcium overload induced by acute ethanol intake and decrease the expression of SPCA1at mRNA and protein level in N2A cell.3. NGF pretreatment increased the expression of SPCA1at mRNA and protein level in N2A cell and alleviated calcium overload induced by acute ethanol intake.Figure17, table12, reference105更多还原