本文選題：參與 + 氧化�。� 參考：《南京醫(yī)科大學(xué)》2007年博士論文

【摘要】： 氧化應(yīng)激(oxidative stress)是機(jī)體最常見的一類應(yīng)激反應(yīng)。在一些損傷因素的作用下，細(xì)胞內(nèi)氧化代謝物增加，或細(xì)胞中抗氧化保護(hù)機(jī)制不足時，使活性氧產(chǎn)生堆積并對細(xì)胞產(chǎn)生毒性，從而產(chǎn)生氧化應(yīng)激。氧化應(yīng)激或者氧自由基的產(chǎn)生已被認(rèn)為是DNA損傷的主要原因之一，可導(dǎo)致一系列的疾病包括癌癥和慢性血管性疾病。堿基切除修復(fù)(base excision repair, BER)是氧化應(yīng)激引起的主要的DNA損傷修復(fù)的形式之一，胞內(nèi)堆積的自由基攻擊DNA造成損傷，從而引起了堿基切除修復(fù)信號通路的激活。 JWA(GenBank: AF070523, 1998)是周建偉等從培養(yǎng)的原代人氣管和支氣管上皮細(xì)胞中分離并克隆的，受ATRA誘導(dǎo)的新的細(xì)胞骨架樣基因。本課題組多年來一直圍繞JWA基因的結(jié)構(gòu)、功能及其相互關(guān)系開展研究工作，取得了一系列原創(chuàng)性有意義的研究成果。先后證實JWA是一種新的微管相關(guān)蛋白(microtubule-associated protein, MAP)，不僅參與全反式維甲酸(ATRA)誘導(dǎo)的細(xì)胞分化調(diào)節(jié)，而且與多種細(xì)胞分化、凋亡誘導(dǎo)劑(如TPA，4HPR和As_2O_3等)的生物學(xué)作用有關(guān)，涉及相應(yīng)的信號通路，而且活躍地參與細(xì)胞對應(yīng)激刺激(如冷應(yīng)激和熱應(yīng)激)的應(yīng)答。近年來許多JWA的同源基因被報道發(fā)現(xiàn)。Ingley等發(fā)現(xiàn)了第一個與JWA同源的基因ARL-6 (AF133912)，兩者氨基酸序列同源性達(dá)93％，提示JWA與ARL-6可能有相似或相近的生物學(xué)功能，而該基因的作用主要涉及PARP與DNA損傷和修復(fù)。 本研究主要應(yīng)用氧化應(yīng)激模型、堿基切除修復(fù)模型和多種分子細(xì)胞生物學(xué)實驗技術(shù)探討JWA參與氧化應(yīng)激的機(jī)制，JWA參與堿基切除修復(fù)信號通途的調(diào)控規(guī)律，其與堿基切除修復(fù)蛋白之間的關(guān)系；初步明確JWA在細(xì)胞應(yīng)答氧化應(yīng)激中發(fā)揮的具體作用，構(gòu)建以JWA參與堿基切除修復(fù)過程為核心的信號轉(zhuǎn)導(dǎo)通路；為最終闡明并完善細(xì)胞氧化應(yīng)激和堿基切除修復(fù)信號通路提供新的視角。 一、胞內(nèi)H_2O_2誘導(dǎo)JWA應(yīng)答氧化應(yīng)激 選取經(jīng)典的氧化應(yīng)激誘導(dǎo)劑H_2O_2和B(a)P處理NIH-3T3和HELF細(xì)胞，隨著處理時間的延長JWA表達(dá)逐漸增加，有明顯的時間依賴效應(yīng)。為了探究何種氧自由基誘導(dǎo)了JWA的表達(dá)，我們將SOD(O_2~-·的拮抗劑)和過氧化氫酶catalase(胞內(nèi)H_2O_2的拮抗劑)應(yīng)用于H_2O_2和B(a)P誘導(dǎo)的氧化應(yīng)激模型中，結(jié)果顯示在NIH-3T3細(xì)胞中用SOD處理后JWA表達(dá)無變化，而用catalase處理后原本被H_2O_2和B(a)P誘導(dǎo)表達(dá)增加的JWA表達(dá)明顯下降，說明胞內(nèi)H_2O_2誘導(dǎo)JWA應(yīng)答氧化應(yīng)激。 二、核因子NFI調(diào)控JWA應(yīng)答氧化應(yīng)激 構(gòu)建了JWA啟動子區(qū)域的系列報告基因質(zhì)粒，利用報告基因試驗我們發(fā)現(xiàn)-107／+107的啟動子區(qū)對氧化應(yīng)激的誘導(dǎo)最為敏感。采用膠泳動率遷移實驗(EMSA)證實有核蛋白和-107／-28啟動子區(qū)域結(jié)合。為了進(jìn)一步揭示此核蛋白的身份，用Southwestern實驗證實此核蛋白的分子量為47kD左右。最后用EMSA的超遷移實驗(supershift)和RNA干涉實驗證實該核蛋白是NFI。由于NFI的結(jié)合基序是CCAAT，而JWA-107／-28啟動子區(qū)包含2個CCAAT反應(yīng)元件，為了確定NFI與JWA近端啟動子區(qū)哪一個CCAAT結(jié)合，我們用Southwestern、EMSA、定點(diǎn)突變和報告基因?qū)嶒炞C實了核因子NFI與JWA近端啟動子區(qū)的第二個CCAAT結(jié)合從而調(diào)控JWA應(yīng)答氧化應(yīng)激。 三、JWA保護(hù)氧化應(yīng)激所誘導(dǎo)的DNA單鏈損傷 成功構(gòu)建了JWA低表達(dá)的細(xì)胞株，采用堿性彗星實驗檢測兩種細(xì)胞對于氧化應(yīng)激誘導(dǎo)下的DNA損傷情況。結(jié)果發(fā)現(xiàn)在B(a)P誘導(dǎo)的過程中，JWA低表達(dá)的細(xì)胞較之對照細(xì)胞DNA損傷程度更加嚴(yán)重，并且修復(fù)的時間延長，說明JWA保護(hù)氧化應(yīng)激所誘導(dǎo)的DNA單鏈損傷 四、JWA與堿基切除修復(fù)蛋白XRCC1和PARP1相互作用 由于JWA能夠保護(hù)氧化應(yīng)激引起的DNA損傷，我們推測JWA可能是直接或間接參與了某個修復(fù)通路，而氧化應(yīng)激引起的主要的修復(fù)途徑是堿基切除修復(fù)，并且堿基切除修復(fù)通路中最重要的信號分子是XRCC1和PARP1。分別用H_2O_2和B(a)P處理質(zhì)粒對照NIH-3T3細(xì)胞和JWA缺陷型NIH-3T3細(xì)胞。在JWA蛋白表達(dá)正常的細(xì)胞中氧化應(yīng)激均能誘導(dǎo)JWA與XRCC1的表達(dá)增加，PARP1的表達(dá)明顯降低；而在JWA蛋白表達(dá)缺陷的細(xì)胞中，氧化應(yīng)激不能誘導(dǎo)XRCC1的表達(dá)變化，但是PARP1的表達(dá)明顯增加。為了進(jìn)一步研究JWA與XRCCI在氧化應(yīng)激誘導(dǎo)的堿基切除修復(fù)中的關(guān)系，采用回復(fù)模型，即將JWA高表達(dá)的質(zhì)粒轉(zhuǎn)染到JWA低表達(dá)的細(xì)胞中，使得JWA的蛋白表達(dá)水平恢復(fù)正常，然后再用H_2O_2和B(a)P處理細(xì)胞，觀察JWA蛋白表達(dá)的變化對XRCC1和PARP1的影響。結(jié)果顯示，當(dāng)采用JWA cDNA pEGFP質(zhì)粒轉(zhuǎn)染JWA缺陷型細(xì)胞，回復(fù)JWA蛋白的正常表達(dá)后，氧化應(yīng)激又能誘導(dǎo)JWA與XRCC1的表達(dá)增加，而PARP1的表達(dá)明顯降低。之后，進(jìn)一步用免疫沉淀和免疫熒光實驗證實了JWA與XRCC1在NIH-3T3細(xì)胞中共定位。說明在氧化應(yīng)激誘導(dǎo)的堿基切除修復(fù)通路中，JWA與堿基切除修復(fù)蛋白XRCC1相互結(jié)合并且表達(dá)一致，而負(fù)調(diào)控PARP1的表達(dá)。 五、JWA在堿基切除修復(fù)信號通路中的定位 分別用H_2O_2和B(a)P處理質(zhì)粒對照NIH-3T3細(xì)胞和JWA缺陷型NIH-3T3細(xì)胞。采用免疫印跡實驗檢測識別蛋白APE1和縫合蛋白LigⅢ的表達(dá)。結(jié)果顯示，，在JWA蛋白表達(dá)正常的細(xì)胞中氧化應(yīng)激均能誘導(dǎo)JWA與LigⅢ的表達(dá)增加，而在JWA蛋白表達(dá)缺陷的細(xì)胞中，氧化應(yīng)激不能誘導(dǎo)LigⅢ的表達(dá)變化；但是JWA對APE1的表達(dá)沒有任何的影響。說明在氧化應(yīng)激過程JWA參與調(diào)控LigⅢ(損傷位點(diǎn)修復(fù)縫合蛋白)而對APE1(損傷位點(diǎn)識別蛋白)無影響。進(jìn)一步說明在堿基切除修復(fù)信號通路中JWA參與了損傷位點(diǎn)的修復(fù)和縫合過程而不參與損傷位點(diǎn)的識別過程。 綜上所述，本研究發(fā)現(xiàn)在氧化應(yīng)激中胞內(nèi)H_2O_2誘導(dǎo)JWA的表達(dá)增加；氧化應(yīng)激誘導(dǎo)核因子NFI結(jié)合到JWA近端啟動子的第二個CCAAT反應(yīng)元件上，從而調(diào)控JWA轉(zhuǎn)錄調(diào)控的增加；被氧化應(yīng)激激活的JWA蛋白通過與堿基切除修復(fù)蛋白XRCC1的結(jié)合，以及負(fù)調(diào)控PARP1的表達(dá)，參與了堿基切除修復(fù)信號通路，保護(hù)細(xì)胞免受了氧化應(yīng)激引起的DNA單鏈損傷。但是JWA在堿基切除修復(fù)中只參與了損傷位點(diǎn)修復(fù)和縫合的過程，沒有參與損傷位點(diǎn)識別的過程。
[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

本文編號：1795217