苯并芘致癌过程中ADP-核糖基化对组蛋白表达的影响

发布时间：2018-06-13 02:50

  本文选题：组蛋白 + ADP-核糖基化　； 参考：《南昌大学》2017年硕士论文

【摘要】：目的:苯并芘(Benzo(a)pyrene,BaP)是目前最为明确的致肺癌化合物,但其致癌分子机制尚未完全阐明。大量文献报道组蛋白的ADP-核糖基化与肿瘤发生密切相关,从组蛋白ADP-核糖基化着手开展BaP致癌作用机理的研究,对于揭示BaP致癌机制具有非常重要的战略性意义。因此,本研究拟通过分析组蛋白ADP-核糖基化修饰在BaP诱导细胞恶性转化过程中的作用,检测和分析BaP作用相关的组蛋白ADP-核糖基化修饰模式,明确ADP-核糖基化在BaP致癌过程中对组蛋白的影响,从而为化学致癌的防治提供新思路。方法:首先选取课题组前期构建的BaP诱导的人支气管上皮细胞(16HBE细胞)恶性转化模型和同样处理的聚-ADP-核糖水解酶(PARG)缺陷的细胞株(shPARG细胞)作为研究对象(处理过程:使用40μmol/L的BaP染毒处理16HBE细胞和PARG缺陷细胞15w),借助透射电子显微镜观察比较两种细胞恶性转化后细胞间期染色质结构的变化,分析BaP诱导细胞转化过程中ADP-核糖基化对组蛋白的可能影响;进一步采用Western blot技术和免疫荧光技术,筛选并鉴定出BaP作用相关的特异性ADP-核糖基化修饰的组蛋白,探讨BaP致癌相关的组蛋白ADP-核糖基化修饰模式;最终通过免疫共沉淀技术分析组蛋白与ADP-核糖间的相互作用,从而明确组蛋白ADP-核糖基化在BaP致癌过程中的可能作用机制。结果:在BaP诱导处理细胞15w后,16HBE细胞内出现核异常,伴有转录活性高的常染色质减少、转录活性很低的异染色质增多,而shPARG细胞内染色质结构改变不明显;Western blot和细胞免疫荧光实验结果表明BaP染毒处理细胞15w后,16HBE细胞内的组蛋白H2A表达明显降低、组蛋白H2B表达显著升高,而shPARG细胞内组蛋白H2A和H2B的表达变化不明显,组蛋白H3和H4在两种细胞内的表达均未见明显改变;进一步使用免疫共沉淀分析结果发现H2A可发生ADP-核糖基化修饰,H2A与PAR间存在相互作用。结论:1、BaP可诱导细胞发生核异常和染色质结构的改变,而PARG基因沉默可对抗细胞内的这些异常;2、在BaP诱导细胞恶性转化过程中,组蛋白H2A可通过发生ADP-核糖基化修饰来维持组蛋白H2A在体内的稳定表达,从而对抗染色质结构异常;3、在BaP致癌过程中,组蛋白可通过发生ADP-核糖基化修饰,以对抗肿瘤的发生。
[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.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114

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本文编号：2012351