JWA参与氧化应激诱导的碱基切除修复信号通路的机制研究

发布时间：2018-04-24 04:41

  本文选题：参与 + 氧化　； 参考：《南京医科大学》2007年博士论文

【摘要】： 氧化应激(oxidative stress)是机体最常见的一类应激反应。在一些损伤因素的作用下，细胞内氧化代谢物增加，或细胞中抗氧化保护机制不足时，使活性氧产生堆积并对细胞产生毒性，从而产生氧化应激。氧化应激或者氧自由基的产生已被认为是DNA损伤的主要原因之一，可导致一系列的疾病包括癌症和慢性血管性疾病。碱基切除修复(base excision repair, BER)是氧化应激引起的主要的DNA损伤修复的形式之一，胞内堆积的自由基攻击DNA造成损伤，从而引起了碱基切除修复信号通路的激活。 JWA(GenBank: AF070523, 1998)是周建伟等从培养的原代人气管和支气管上皮细胞中分离并克隆的，受ATRA诱导的新的细胞骨架样基因。本课题组多年来一直围绕JWA基因的结构、功能及其相互关系开展研究工作，取得了一系列原创性有意义的研究成果。先后证实JWA是一种新的微管相关蛋白(microtubule-associated protein, MAP)，不仅参与全反式维甲酸(ATRA)诱导的细胞分化调节，而且与多种细胞分化、凋亡诱导剂(如TPA，4HPR和As_2O_3等)的生物学作用有关，涉及相应的信号通路，而且活跃地参与细胞对应激刺激(如冷应激和热应激)的应答。近年来许多JWA的同源基因被报道发现。Ingley等发现了第一个与JWA同源的基因ARL-6 (AF133912)，两者氨基酸序列同源性达93％，提示JWA与ARL-6可能有相似或相近的生物学功能，而该基因的作用主要涉及PARP与DNA损伤和修复。 本研究主要应用氧化应激模型、碱基切除修复模型和多种分子细胞生物学实验技术探讨JWA参与氧化应激的机制，JWA参与碱基切除修复信号通途的调控规律，其与碱基切除修复蛋白之间的关系；初步明确JWA在细胞应答氧化应激中发挥的具体作用，构建以JWA参与碱基切除修复过程为核心的信号转导通路；为最终阐明并完善细胞氧化应激和碱基切除修复信号通路提供新的视角。 一、胞内H_2O_2诱导JWA应答氧化应激 选取经典的氧化应激诱导剂H_2O_2和B(a)P处理NIH-3T3和HELF细胞，随着处理时间的延长JWA表达逐渐增加，有明显的时间依赖效应。为了探究何种氧自由基诱导了JWA的表达，我们将SOD(O_2~-·的拮抗剂)和过氧化氢酶catalase(胞内H_2O_2的拮抗剂)应用于H_2O_2和B(a)P诱导的氧化应激模型中，结果显示在NIH-3T3细胞中用SOD处理后JWA表达无变化，而用catalase处理后原本被H_2O_2和B(a)P诱导表达增加的JWA表达明显下降，说明胞内H_2O_2诱导JWA应答氧化应激。 二、核因子NFI调控JWA应答氧化应激 构建了JWA启动子区域的系列报告基因质粒，利用报告基因试验我们发现-107／+107的启动子区对氧化应激的诱导最为敏感。采用胶泳动率迁移实验(EMSA)证实有核蛋白和-107／-28启动子区域结合。为了进一步揭示此核蛋白的身份，用Southwestern实验证实此核蛋白的分子量为47kD左右。最后用EMSA的超迁移实验(supershift)和RNA干涉实验证实该核蛋白是NFI。由于NFI的结合基序是CCAAT，而JWA-107／-28启动子区包含2个CCAAT反应元件，为了确定NFI与JWA近端启动子区哪一个CCAAT结合，我们用Southwestern、EMSA、定点突变和报告基因实验证实了核因子NFI与JWA近端启动子区的第二个CCAAT结合从而调控JWA应答氧化应激。 三、JWA保护氧化应激所诱导的DNA单链损伤 成功构建了JWA低表达的细胞株，采用碱性彗星实验检测两种细胞对于氧化应激诱导下的DNA损伤情况。结果发现在B(a)P诱导的过程中，JWA低表达的细胞较之对照细胞DNA损伤程度更加严重，并且修复的时间延长，说明JWA保护氧化应激所诱导的DNA单链损伤 四、JWA与碱基切除修复蛋白XRCC1和PARP1相互作用 由于JWA能够保护氧化应激引起的DNA损伤，我们推测JWA可能是直接或间接参与了某个修复通路，而氧化应激引起的主要的修复途径是碱基切除修复，并且碱基切除修复通路中最重要的信号分子是XRCC1和PARP1。分别用H_2O_2和B(a)P处理质粒对照NIH-3T3细胞和JWA缺陷型NIH-3T3细胞。在JWA蛋白表达正常的细胞中氧化应激均能诱导JWA与XRCC1的表达增加，PARP1的表达明显降低；而在JWA蛋白表达缺陷的细胞中，氧化应激不能诱导XRCC1的表达变化，但是PARP1的表达明显增加。为了进一步研究JWA与XRCCI在氧化应激诱导的碱基切除修复中的关系，采用回复模型，即将JWA高表达的质粒转染到JWA低表达的细胞中，使得JWA的蛋白表达水平恢复正常，然后再用H_2O_2和B(a)P处理细胞，观察JWA蛋白表达的变化对XRCC1和PARP1的影响。结果显示，当采用JWA cDNA pEGFP质粒转染JWA缺陷型细胞，回复JWA蛋白的正常表达后，氧化应激又能诱导JWA与XRCC1的表达增加，而PARP1的表达明显降低。之后，进一步用免疫沉淀和免疫荧光实验证实了JWA与XRCC1在NIH-3T3细胞中共定位。说明在氧化应激诱导的碱基切除修复通路中，JWA与碱基切除修复蛋白XRCC1相互结合并且表达一致，而负调控PARP1的表达。 五、JWA在碱基切除修复信号通路中的定位 分别用H_2O_2和B(a)P处理质粒对照NIH-3T3细胞和JWA缺陷型NIH-3T3细胞。采用免疫印迹实验检测识别蛋白APE1和缝合蛋白LigⅢ的表达。结果显示，，在JWA蛋白表达正常的细胞中氧化应激均能诱导JWA与LigⅢ的表达增加，而在JWA蛋白表达缺陷的细胞中，氧化应激不能诱导LigⅢ的表达变化；但是JWA对APE1的表达没有任何的影响。说明在氧化应激过程JWA参与调控LigⅢ(损伤位点修复缝合蛋白)而对APE1(损伤位点识别蛋白)无影响。进一步说明在碱基切除修复信号通路中JWA参与了损伤位点的修复和缝合过程而不参与损伤位点的识别过程。 综上所述，本研究发现在氧化应激中胞内H_2O_2诱导JWA的表达增加；氧化应激诱导核因子NFI结合到JWA近端启动子的第二个CCAAT反应元件上，从而调控JWA转录调控的增加；被氧化应激激活的JWA蛋白通过与碱基切除修复蛋白XRCC1的结合，以及负调控PARP1的表达，参与了碱基切除修复信号通路，保护细胞免受了氧化应激引起的DNA单链损伤。但是JWA在碱基切除修复中只参与了损伤位点修复和缝合的过程，没有参与损伤位点识别的过程。
[Abstract]:Oxidative stress is the most common type of stress reaction in the organism . Under the action of some damage factors , the oxidative stress , oxidative stress , or oxygen free radical production have been considered as one of the main causes of DNA damage , which can lead to a series of diseases including cancer and chronic vascular disease . Base excision repair ( BER ) is one of the main forms of DNA damage repair caused by oxidative stress . Base excision repair ( BER ) is one of the main causes of DNA damage caused by oxidative stress .






JWA ( GenBank : AF070523 , 1998 ) is a new cytoskeletal - like gene isolated and cloned from cultured primary human trachea and bronchial epithelial cells , which is induced by ATRA .






This study mainly applies oxidative stress model , base excision repair model and various molecular cytobiology experimental techniques to explore the mechanism of JWA ' s participation in oxidative stress . JWA is involved in the regulation and regulation of the pathway of the repair signal , which is related to the base excision repair protein ;
The specific role of JWA in cell response oxidative stress was preliminarily defined , and signal transduction pathways were constructed using JWA as the core .
To provide a new visual angle for the final clarification and improvement of cell oxidative stress and base excision repair signal pathway .






I , H _ 2O _ 2 - induced oxidative stress in JWA






In order to investigate the expression of JWA induced by H _ 2O _ 2 and B ( a ) P in NIH - 3T3 cells , the results showed that the expression of JWA was not changed after SOD treatment in NIH - 3T3 cells .






II . Nuclear factor NFI regulates JWA to respond to oxidative stress






In order to further reveal the identity of NFI and JWA - 107 / -28 promoter region , we confirmed that the nuclear protein was NFI . In order to find out which CCAAT binding motif in NFI and JWA near - end promoter region , we demonstrated that NFI combined with the second CCAAT of JWA near - end promoter region to regulate JWA response oxidative stress .






III . Single - stranded DNA Damage Induced by Oxidative Stress by JWA






A low expression of JWA was successfully constructed , and the DNA damage induced by oxidative stress was detected by alkaline comet assay .






IV . Interaction between JWA and base excision repair protein xrCC1 and PARP1






Because JWA was able to protect DNA damage induced by oxidative stress , we speculated that JWA might be directly or indirectly involved in a repair pathway , while the major repair pathway caused by oxidative stress was base excision repair , and the most important signal molecules in the base excision repair pathway were the expression of the plasmid control NIH - 3T3 cells and JWA - deficient NIH - 3T3 cells .
In order to further study the relationship between JWA gene expression level and JWA protein expression level , JWA was transfected into low - expression cells of JWA , and the expression of JWA protein was significantly decreased .






5 . Location of JWA in the Repair Signal Transduction Pathway of Base excision






NIH - 3T3 cells and NIH - 3T3 cells were treated with H _ 2O _ 2 and B ( a ) P respectively . The expression of APE1 and Lig 鈪

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