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
17-23
,共7页
武小媛%曲丽艳%全康%蒋燕灵%唐修文
武小媛%麯麗豔%全康%蔣燕靈%唐脩文
무소원%곡려염%전강%장연령%당수문
氢醌类/药理学%硫氰酸盐类/药理学%Caco-2细胞%结肠肿瘤%转录因子%基因表达%反应元件%核胞浆转运蛋白类
氫醌類/藥理學%硫氰痠鹽類/藥理學%Caco-2細胞%結腸腫瘤%轉錄因子%基因錶達%反應元件%覈胞漿轉運蛋白類
경곤류/약이학%류청산염류/약이학%Caco-2세포%결장종류%전록인자%기인표체%반응원건%핵포장전운단백류
Hydroquinones/pharmacol%Hydroquinones/pharmacol%Caco-2 cells%Colonic neoplasms%Transcription factors%Gene expression%Response elements%Karyopherins
目的:研究抗氧化性诱导剂(激活剂)叔丁基对苯二酚(tert-butylhydroquinone,tBHQ)和莱菔硫烷[1-异硫氰酸-(4R,S)-(甲基亚磺酰基)丁烷](sulforaphane,SFN)对人结肠癌细胞株Caco2的Nrf2-ARE信号通路的影响.方法:选取不同的时间点,分别用20 μmol/L tBHQ和5 μmol/L SFN处理Caco2细胞,从蛋白质水平、mRNA水平和蛋白质定位3个方面,分别用Western blotting、real-time PCR和免疫荧光染色(immunoflourescence staining,IF)方法来检测Nrf2以及其调控基因AKR1C1和NQO1(Western blotting,real-time PCR)表达的变化.结果:细胞核中Nrf2基因表达的最佳时间点为加药诱导后8 h,细胞质中Nrf2调控基因AKR1C1和NQO1表达的最佳时间点为加药诱导后16 h.tBHQ的诱导作用在撤去之后仍能持续4 h.结论:本研究使用激活剂tBHQ和SFN对Caco2细胞的Nrf2-ARE信号通路进行诱导,取得了最佳的诱导时间点,并研究了撤去tBHQ后持续诱导时间,为今后在肿瘤化学预防研究中,筛选合适的激活剂及其剂量和使用次数提供了重要依据,也为研究Nrf2-ARE信号通路的抑制剂提供了重要信息.
目的:研究抗氧化性誘導劑(激活劑)叔丁基對苯二酚(tert-butylhydroquinone,tBHQ)和萊菔硫烷[1-異硫氰痠-(4R,S)-(甲基亞磺酰基)丁烷](sulforaphane,SFN)對人結腸癌細胞株Caco2的Nrf2-ARE信號通路的影響.方法:選取不同的時間點,分彆用20 μmol/L tBHQ和5 μmol/L SFN處理Caco2細胞,從蛋白質水平、mRNA水平和蛋白質定位3箇方麵,分彆用Western blotting、real-time PCR和免疫熒光染色(immunoflourescence staining,IF)方法來檢測Nrf2以及其調控基因AKR1C1和NQO1(Western blotting,real-time PCR)錶達的變化.結果:細胞覈中Nrf2基因錶達的最佳時間點為加藥誘導後8 h,細胞質中Nrf2調控基因AKR1C1和NQO1錶達的最佳時間點為加藥誘導後16 h.tBHQ的誘導作用在撤去之後仍能持續4 h.結論:本研究使用激活劑tBHQ和SFN對Caco2細胞的Nrf2-ARE信號通路進行誘導,取得瞭最佳的誘導時間點,併研究瞭撤去tBHQ後持續誘導時間,為今後在腫瘤化學預防研究中,篩選閤適的激活劑及其劑量和使用次數提供瞭重要依據,也為研究Nrf2-ARE信號通路的抑製劑提供瞭重要信息.
목적:연구항양화성유도제(격활제)숙정기대분이분(tert-butylhydroquinone,tBHQ)화래복류완[1-이류청산-(4R,S)-(갑기아광선기)정완](sulforaphane,SFN)대인결장암세포주Caco2적Nrf2-ARE신호통로적영향.방법:선취불동적시간점,분별용20 μmol/L tBHQ화5 μmol/L SFN처리Caco2세포,종단백질수평、mRNA수평화단백질정위3개방면,분별용Western blotting、real-time PCR화면역형광염색(immunoflourescence staining,IF)방법래검측Nrf2이급기조공기인AKR1C1화NQO1(Western blotting,real-time PCR)표체적변화.결과:세포핵중Nrf2기인표체적최가시간점위가약유도후8 h,세포질중Nrf2조공기인AKR1C1화NQO1표체적최가시간점위가약유도후16 h.tBHQ적유도작용재철거지후잉능지속4 h.결론:본연구사용격활제tBHQ화SFN대Caco2세포적Nrf2-ARE신호통로진행유도,취득료최가적유도시간점,병연구료철거tBHQ후지속유도시간,위금후재종류화학예방연구중,사선합괄적격활제급기제량화사용차수제공료중요의거,야위연구Nrf2-ARE신호통로적억제제제공료중요신식.
Objective: To investigate the effect of tBHQ and sulforaphane on the protein expression in Nrf2-ARE signaling pathway of Caco2 cells.Methods: Human colorectal carcinoma Caco2 cells were treated with 20 μmol/L tBHQ and 5 μmol/L sulforaphane (SFN) respectively.Real time PCR,Western blotting and immunoflourescence staining (IF) were performed to measure the target gene expression.Results:Nrf2,AKR1C1 and NQO1 protein expressions were increased time-dependently in Caco2 cells after treatment with tBHQ and SFN.Time-course experiments showed that tBHQ and SFN increased the accumulation of Nrf2,and concomitantly increased the protein levels of AKR1C1 and NQO1.Real-time PCR and Western blotting showed that tBHQ and SFN significantly increased the expression of Nrf2 at 8h after the treatment,and AKR1C1 and NQO1 at 16 h.Confocal microscopy technique showed that Nrf2 accumulated in the nucleus at 6-8 h after treatment with tBHQ.After 1 h treatment with tBHQ the nuclear Nrf2 maintained at elevated level for at least 4 h with tBHQ withdrawn.Conclusions: tBHQ and SFN induced nuclear accumulation of Nrf2 and activated Nrf2-dependent regulation of ARE-mediated gene expression in Caco2 cells.In addition,the results provide experimental evidence for choosing the dose and frequency of the inducer in cancer chemoprevention study and in developing inhibitors of Nrf2-ARE signaling pathway.
