浙江大学学报（医学版）
浙江大學學報（醫學版）
절강대학학보（의학판）
JOURNAL OF ZHEJIANG UNIVERSITY MEDICAL SCIENCES
2010年
1期
24-29
,共6页
核胞浆转运蛋白类%反应元件%癌%非小细胞肺%腺嘌呤核苷酸类%咪唑类%基因表达
覈胞漿轉運蛋白類%反應元件%癌%非小細胞肺%腺嘌呤覈苷痠類%咪唑類%基因錶達
핵포장전운단백류%반응원건%암%비소세포폐%선표령핵감산류%미서류%기인표체
Karyopherins%Response elements%Carcinoma,non-small-cell lung%Adenine nucleoti-des%Imidazoles%Gene expression
目的:利用细胞核亚细胞器结构的一些特异性蛋白质SC35、核孔复合体(nuclear pore complex,NPC)、RNA聚合酶Ⅱ(PolⅡ)及 Y12等标记亚细胞器,探讨转录抑制剂5,6-dichloro-1-β(-D)-ribofuranosylbenzimidazole (DRB)与α-Amanitin对人非小细胞肺癌A549细胞中Nrf2的表达与定位的影响. 方法:①50 mg/L DRB和2.5 mg/L α-Amanitin分别作用于A549细胞1 h和6 h后,Western blotting检测Nrf2及其相关蛋白的表达变化.②50 mg/L DRB和2.5 mg/L α-Amanitin分别处理A549细胞1 h,利用免疫荧光细胞技术检测Nrf2、SC35、NPC、RNA PolⅡ、Y12形态结构的变化,并研究Nrf2定位的变化.结果:①与对照组相比,DRB和α-Amanitin作用1 h后, Nrf2及其调控的下游基因HO-1、AKR1C和NQO1的蛋白质表达变化不明显,但6 h作用后,这些蛋白质的表达明显降低.②激光共聚焦显微镜观察显示,细胞经DRB和α-Amanitin处理1 h后,SC35的荧光强度明显增强,PolⅡ的荧光强度减弱,而Nrf2的定位无明显改变;NPC、Y12的表达无明显变化,且与Nrf2无共定位现象.结论:DRB和α-Amanitin能抑制Nrf2及其HO-1、AKR1C和NQO1的表达,但是对Nrf2的定位没有显著影响.
目的:利用細胞覈亞細胞器結構的一些特異性蛋白質SC35、覈孔複閤體(nuclear pore complex,NPC)、RNA聚閤酶Ⅱ(PolⅡ)及 Y12等標記亞細胞器,探討轉錄抑製劑5,6-dichloro-1-β(-D)-ribofuranosylbenzimidazole (DRB)與α-Amanitin對人非小細胞肺癌A549細胞中Nrf2的錶達與定位的影響. 方法:①50 mg/L DRB和2.5 mg/L α-Amanitin分彆作用于A549細胞1 h和6 h後,Western blotting檢測Nrf2及其相關蛋白的錶達變化.②50 mg/L DRB和2.5 mg/L α-Amanitin分彆處理A549細胞1 h,利用免疫熒光細胞技術檢測Nrf2、SC35、NPC、RNA PolⅡ、Y12形態結構的變化,併研究Nrf2定位的變化.結果:①與對照組相比,DRB和α-Amanitin作用1 h後, Nrf2及其調控的下遊基因HO-1、AKR1C和NQO1的蛋白質錶達變化不明顯,但6 h作用後,這些蛋白質的錶達明顯降低.②激光共聚焦顯微鏡觀察顯示,細胞經DRB和α-Amanitin處理1 h後,SC35的熒光彊度明顯增彊,PolⅡ的熒光彊度減弱,而Nrf2的定位無明顯改變;NPC、Y12的錶達無明顯變化,且與Nrf2無共定位現象.結論:DRB和α-Amanitin能抑製Nrf2及其HO-1、AKR1C和NQO1的錶達,但是對Nrf2的定位沒有顯著影響.
목적:이용세포핵아세포기결구적일사특이성단백질SC35、핵공복합체(nuclear pore complex,NPC)、RNA취합매Ⅱ(PolⅡ)급 Y12등표기아세포기,탐토전록억제제5,6-dichloro-1-β(-D)-ribofuranosylbenzimidazole (DRB)여α-Amanitin대인비소세포폐암A549세포중Nrf2적표체여정위적영향. 방법:①50 mg/L DRB화2.5 mg/L α-Amanitin분별작용우A549세포1 h화6 h후,Western blotting검측Nrf2급기상관단백적표체변화.②50 mg/L DRB화2.5 mg/L α-Amanitin분별처리A549세포1 h,이용면역형광세포기술검측Nrf2、SC35、NPC、RNA PolⅡ、Y12형태결구적변화,병연구Nrf2정위적변화.결과:①여대조조상비,DRB화α-Amanitin작용1 h후, Nrf2급기조공적하유기인HO-1、AKR1C화NQO1적단백질표체변화불명현,단6 h작용후,저사단백질적표체명현강저.②격광공취초현미경관찰현시,세포경DRB화α-Amanitin처리1 h후,SC35적형광강도명현증강,PolⅡ적형광강도감약,이Nrf2적정위무명현개변;NPC、Y12적표체무명현변화,차여Nrf2무공정위현상.결론:DRB화α-Amanitin능억제Nrf2급기HO-1、AKR1C화NQO1적표체,단시대Nrf2적정위몰유현저영향.
Objective: To investigate the effects of transcriptional inhibitors 5,6-dichloro-1-b-D- ribofuranosylbenzimidazole (DRB) and α-Amanitin on the localization of Nrf2 in the nucleus.Methods:A549 cells were treated with DRB(50 mg/L)or α-Amanitin(2.5 mg/L)for 1 h and 6 h in serum-free medium,respectively.The expressions of Nrf2,HO-1,NQO1 and AKR1C were detected by Western blotting analysis.The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or α-Amanitin for 1 h.Results: The expressions of Nrf2 and Nrf2-ARE gene batteries HO-1,AKR1C and NQO1 were decreased after 6 h treated with either DRB or α-Amanitin.The expression of SC35 was up-regulated but RNA PolⅡ was down-regulated; Y12 and NPC did not significantly change.The localization of Nrf2 in the cell nucleus did not change significantly.Conclusion:DRB and α-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1,AKR1C and NQO1,but may have no effect on the localization of Nrf2.
