本文選題：組蛋白 + ADP-核糖基化　； 參考：《南昌大學》2017年碩士論文

【摘要】：目的:苯并芘(Benzo(a)pyrene,BaP)是目前最為明確的致肺癌化合物,但其致癌分子機制尚未完全闡明。大量文獻報道組蛋白的ADP-核糖基化與腫瘤發(fā)生密切相關(guān),從組蛋白ADP-核糖基化著手開展BaP致癌作用機理的研究,對于揭示BaP致癌機制具有非常重要的戰(zhàn)略性意義。因此,本研究擬通過分析組蛋白ADP-核糖基化修飾在BaP誘導細胞惡性轉(zhuǎn)化過程中的作用,檢測和分析BaP作用相關(guān)的組蛋白ADP-核糖基化修飾模式,明確ADP-核糖基化在BaP致癌過程中對組蛋白的影響,從而為化學致癌的防治提供新思路。方法:首先選取課題組前期構(gòu)建的BaP誘導的人支氣管上皮細胞(16HBE細胞)惡性轉(zhuǎn)化模型和同樣處理的聚-ADP-核糖水解酶(PARG)缺陷的細胞株(shPARG細胞)作為研究對象(處理過程:使用40μmol/L的BaP染毒處理16HBE細胞和PARG缺陷細胞15w),借助透射電子顯微鏡觀察比較兩種細胞惡性轉(zhuǎn)化后細胞間期染色質(zhì)結(jié)構(gòu)的變化,分析BaP誘導細胞轉(zhuǎn)化過程中ADP-核糖基化對組蛋白的可能影響;進一步采用Western blot技術(shù)和免疫熒光技術(shù),篩選并鑒定出BaP作用相關(guān)的特異性ADP-核糖基化修飾的組蛋白,探討B(tài)aP致癌相關(guān)的組蛋白ADP-核糖基化修飾模式;最終通過免疫共沉淀技術(shù)分析組蛋白與ADP-核糖間的相互作用,從而明確組蛋白ADP-核糖基化在BaP致癌過程中的可能作用機制。結(jié)果:在BaP誘導處理細胞15w后,16HBE細胞內(nèi)出現(xiàn)核異常,伴有轉(zhuǎn)錄活性高的常染色質(zhì)減少、轉(zhuǎn)錄活性很低的異染色質(zhì)增多,而shPARG細胞內(nèi)染色質(zhì)結(jié)構(gòu)改變不明顯;Western blot和細胞免疫熒光實驗結(jié)果表明BaP染毒處理細胞15w后,16HBE細胞內(nèi)的組蛋白H2A表達明顯降低、組蛋白H2B表達顯著升高,而shPARG細胞內(nèi)組蛋白H2A和H2B的表達變化不明顯,組蛋白H3和H4在兩種細胞內(nèi)的表達均未見明顯改變;進一步使用免疫共沉淀分析結(jié)果發(fā)現(xiàn)H2A可發(fā)生ADP-核糖基化修飾,H2A與PAR間存在相互作用。結(jié)論:1、BaP可誘導細胞發(fā)生核異常和染色質(zhì)結(jié)構(gòu)的改變,而PARG基因沉默可對抗細胞內(nèi)的這些異常;2、在BaP誘導細胞惡性轉(zhuǎn)化過程中,組蛋白H2A可通過發(fā)生ADP-核糖基化修飾來維持組蛋白H2A在體內(nèi)的穩(wěn)定表達,從而對抗染色質(zhì)結(jié)構(gòu)異常;3、在BaP致癌過程中,組蛋白可通過發(fā)生ADP-核糖基化修飾,以對抗腫瘤的發(fā)生。
[Abstract]:Objective: Benzoopyrene Benzoa pyrene BaPe is the most definite compound of lung cancer, but the molecular mechanism of its carcinogenesis has not been fully elucidated. A large number of literatures have reported that ADP- ribonucleylation of histone is closely related to tumorigenesis. It is very important to study the carcinogenic mechanism of bap from histone ADP- ribonucleylation, which is of great strategic significance in revealing the carcinogenic mechanism of bap. Therefore, by analyzing the role of histone ADP- ribosylation modification in the process of malignant transformation induced by bap, we detected and analyzed the histone ADP- ribosylation modification model related to the role of bap. The effect of ADP- ribosylation on histone in the carcinogenesis of bap was clarified, thus providing a new idea for the prevention and treatment of chemical carcinogenesis. Methods: the malignant transformation model of bap induced human bronchial epithelial cell line (bap) and the same treated cell line of Poly (ADP- ribonucleohydrolase) deficient cell line (shPARG cell line) were selected. Process: 16HBE cells and PARG-deficient cells were treated with 40 渭 mol / L bap for 15 weeks. The chromatin structure of the two cells after malignant transformation was observed and compared by transmission electron microscopy. The possible effects of ADP- ribonylation on histone in the process of cell transformation induced by bap were analyzed, and the specific ADP- ribonylation modified histones were screened and identified by Western blot and immunofluorescence techniques. To explore the histone ADP- ribosylation modification model related to the carcinogenesis of bap, and finally to analyze the interaction between histone and ADP- ribose by immunoprecipitation, so as to clarify the possible mechanism of histone ADP- ribonylation in the carcinogenesis of bap. Results: after 15 weeks of treatment with bap, there were nuclear abnormalities in 16HBE cells, accompanied by the decrease of normal chromatin with high transcriptional activity and the increase of heterochromatin with very low transcriptional activity. The results of Western blot and cellular immunofluorescence assay showed that the expression of histone H2A was significantly decreased and the expression of histone H2B was significantly increased in the cells treated with bap for 15 weeks. However, the expression of histone H2A and H2B in shPARG cells did not change significantly, but the expression of histone H3 and H4 in both cells did not change significantly. The results of immunoprecipitation analysis showed that the interaction between ADP- ribosylation modified H2A and par could occur. Conclusion the cell nuclear abnormality and chromatin structure change can be induced by 1: 1 Bap, and parg gene silencing can antagonize these aberrations in the cell, and in the process of malignant transformation induced by bap, PARG gene silencing can antagonize these abnormalities. Histone H2A can maintain the stable expression of histone H2A in vivo through ADP- ribosylation modification, which can counteract chromatin structural abnormalities. During the carcinogenesis of bap, histone H2A can be modified by ADP- ribosylation to prevent the occurrence of tumor.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114

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本文編號：2012351