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  本文關鍵詞： 酒精 SPCA1 高爾基體 Ca~(2+) NAC NGF　出處：《中南大學》2013年博士論文　論文類型：學位論文

【摘要】：目的： 本論文研究旨在探索氧化應激是否介導酒精對高爾基體SPCAl的影響,以及神經(jīng)生長因子是否具備改善酒精毒性的作用,為酒精相關疾病治療提供新思路。 方法： 1.培養(yǎng)小鼠神經(jīng)細胞瘤N2A細胞,建立酒精封閉系統(tǒng)； 2.實驗分三個模塊：急性酒精作用模塊、抗氧化劑NAC預處理模塊、神經(jīng)生長因子NGF預處理模塊； 3.光學顯微鏡、MTT法評價急性酒精作用對N2A細胞生存的影響； 4.熒光顯微鏡、流式細胞儀觀察及檢測細胞內(nèi)氧化應激水平； 5.多功能酶標儀檢測細胞內(nèi)游離鈣離子濃度的變化； 6.實時熒光定量PCR檢測基因ATP2C1表達變化； 7. Western blot檢測蛋白SPCAl表達變化。 結果： 1.急性酒精作用后,小鼠N2A細胞形態(tài)結構無明顯變化,MTT吸光度值變化無統(tǒng)計學差異,說明酒精200mM濃度以下24h作用對小鼠神經(jīng)細胞瘤N2A細胞生存無明顯影響； 2.急性酒精作用后,各濃度酒精亞組均較空白組細胞熒光強度高,且隨酒精濃度上升逐漸增大,在酒精100mM濃度熒光強度增幅最大(16.3%),隨后小幅下降。NAC預處理后,NAC1mM、2mM濃度下細胞內(nèi)氧化應激相比單獨酒精作用無明顯變化,僅在NAC4mM濃度下細胞內(nèi)氧化應激較單獨酒精作用組低,降幅為3.7%。 3.急性酒精作用后,各濃度酒精亞組均較空白組細胞內(nèi)游離鈣離子濃度增高,且隨酒精濃度上升逐漸增大,在100mM濃度時增幅最大(22.4%),隨后小幅下降。NAC預處理后,各濃度NAC亞組細胞內(nèi)游離鈣離子濃度均較單獨酒精作用組明顯降低,且下降程度隨NAC濃度上升逐漸增大,在4mM濃度降幅最大(31.8%)。NGF預處理后,各濃度NGF亞組的細胞內(nèi)游離鈣離子濃度均較單獨酒精作用組明顯降低,且下降程度隨NGF濃度上升逐漸增大,在50ng/ml濃度時降幅最大(31.4%),隨后小幅下降。 4.急性酒精作用后,各濃度酒精亞組基因ATP2C1、蛋白SPCA1表達水平均較空白組升高,且隨酒精濃度上升逐漸增大,在200mM濃度時達最大增幅(45%,34.9%)。NAC預處理后,各濃度NAC亞組基因ATP2C1、蛋白SPCA1表達水平均較單獨酒精作用亞組下降,且下降程度隨NAC濃度升高而增大,在4mM濃度下降幅度最大(30%,12%)。NGF預處理后,各濃度NGF亞組基因ATP2C1、蛋白SPCA1表達水平均較單獨酒精作用亞組升高,且上升程度隨NGF濃度升高而增大,在100ng/ml濃度時表達最高(16%,6%)。 結論： 1.急性酒精作用導致了細胞內(nèi)氧化應激、鈣超載,上調(diào)了SPCA1的表達。 2.NAC預處理可緩解急性酒精所致細胞內(nèi)氧化應激、鈣超載,下調(diào)SPCAl表達。 3.NGF預處理可上調(diào)SPCAl表達,緩解急性酒精所致細胞內(nèi)鈣超載。圖17幅,表12張,參考文獻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.
【學位授予單位】：中南大學
【學位級別】：博士
【學位授予年份】：2013
【分類號】：R749.62

【參考文獻】

相關期刊論文 前3條

1 殷憲敏,楊明峰,李睿,陳昌偉,劉瑞瑣;幾種不同藥物對缺氧缺血新生大鼠神經(jīng)元凋亡的影響[J];泰山醫(yī)學院學報;2003年01期

2 羅有同,肖昕;N-乙酰半胱氨酸對缺氧缺血性腦病新生鼠腦細胞凋亡的干預效果[J];武警醫(yī)學;2004年09期

3 劉霞;李長齡;尹廣宇;權龍浩;;N-乙酰半胱氨酸對乙醇代謝的影響及機制研究[J];中國藥學雜志;2006年14期

，

本文編號：1521363