【正文】 the cells were treated for 60 min (A), the relative fluorescence intensity were measured by a microplate reader(B). Each rectangle represents the mean 177。SE of three replicates (n=3). Compared with CK. differences were significant at P (*) and P (**) and pared with 10%E, differences were significant at P () and P (), according to ttest.ROS的種類很多，其中常見的為OH2O2等?；钚匝醯姆N類不同，其起作用的方式也不同，本實驗分別以熒光染料DHE和DCFHDA為探針，測定了不同處理酵母中的O2和H2O2的水平。 GSH，Met和NAC對乙醇脅迫釀酒酵母胞內(nèi)O2含量的影響。培養(yǎng)至對數(shù)期的酵母分別在含H2O、10% ethanol、10% ethanol and mM GSH、10% ethanol and 6 mM Met、10% ethanol and 10 mM NAC的YPD培養(yǎng)基中生長1h，DHE染色10 min。熒光顯微鏡所拍照片(A)。熒光酶標儀所測熒光強度(B)。所有值均為三次平行實驗的平均值177。標準誤（n=3）。使用SPSS進行T檢驗，與CK相比，t ：差異顯著(*)，t ：差異極顯著(**) ；與10%E相比，t ：差異顯著()，t ：差異極顯著()。. Effects of GSH、Met and NAC on the O2 level of cells under ethanol stress. Cells were grown in YPD medium until midlog phase and treated with indicated reagents for 1h. Cells were stained with DHE, fluorescent micrographs of the cells were taken under a fluorescence microscope(A). The relative fluorescence intensity were measured by a microplate reader(B). CK: control, 10%E: 10% ethanol, 10%E+GSH: 10% ethanol and mM GSH, 10%E+Met: 10% ethanol and 6 mM Met, 10%E+NAC: 10% ethanol and 10 mM NAC. Each rectangle represents the mean 177。SE of three replicates (n=3). Compared with CK, differences were significant at t (*) and t (**) and pared with 10%E, differences were significant at t () and t (), according to ttest. O2雖然活性不強，但可以直接與某些蛋白發(fā)生反應，造成蛋白損傷；與羥基（OH）結(jié)合后的產(chǎn)物還會導致細胞DNA損壞，破壞細胞機體功能。，乙醇可以明顯提高DHE染色后細胞的熒光強度，分別加入GSH、Met和NAC后發(fā)現(xiàn)三種化合物都可以降低乙醇增加的紅色熒光強度，進一步用熒光酶標儀驗證，發(fā)現(xiàn)GSH、Met 和NAC確實可以非常明顯的降低乙醇增加的紅色熒光強度，但是仍比對照組的水平要高。以上結(jié)果表明乙醇處理增加了細胞內(nèi)O2的水平，而GSH、Met 和NAC可以恢復乙醇增加的O2含量。 GSH，Met和NAC對乙醇脅迫釀酒酵母細胞O2含量的影響。培養(yǎng)至對數(shù)期的酵母分別在含H2O、10% ethanol、10% ethanol 和 mM GSH、10% ethanol和 6 mM Met、10% ethanol 和10 mM NAC的YPD培養(yǎng)基中生長1 h ，NBT染色。光學顯微鏡所拍照片(A)。酶標儀所測吸光度值(B)。所有值均為三次平行實驗的平均值177。標準誤（n=3）。使用SPSS進行T檢驗，與CK相比，t ：差異顯著(*)，P ：差異極顯著(**) 。與10%E相比，t ：差異顯著()，t ：差異極顯著()。. Effects of GSH、Met and NAC on the O2 level of cells under ethanol stress. Cell were grown in YPD medium until midlog phase and treated with indicated reagents for 1 h. A）Cells were stained with NBT, micrographs of the cells were taken under a microscope. B) Cells were stained with NBT, the relative intensity were measured by a microplate reader. CK: control, 10%E: 10% ethanol, 10%E+GSH: 10% ethanol and mM GSH, 10%E+Met: 10% ethanol and 6 mM Met, 10%E+NAC: 10% ethanol and 10 mM NAC. Each rectangle represents the mean 177。SE of three replicates (n=3). Compared with CK, differences were significant at t (*) and t (**) and pared with 10%E, differences were significant at t () and t (), according to ttest. NBT (pNitroblue tetrazolium)可以和O2反應形成藍紫色的甲瓚，其顏色深淺可以反映O2產(chǎn)生的多少。進一步用NBT法驗證GSH、Met和NAC對乙醇脅迫的釀酒酵母中O2含量的影響，與用熒光探針DCFHDA檢測一致，10%乙醇處理60 min的釀酒酵母中O2明顯提高，但同時分別加入GSH、Met和NAC后，發(fā)現(xiàn)只有Met能有效的降低乙醇提高的O2含量。兩種方法結(jié)果不一致，有可能是因為DCFHDA和NBT對O2的敏感性不同造成的。 GSH、Met和NAC對乙醇脅迫釀酒酵母細胞H2O2含量的影響。培養(yǎng)至對數(shù)期的酵母分別在含0、10% ethanol、10% ethanol+ mM GSH、10% ethanol +6 mM Met、10%ethanol+10 mM NAC的YPD培養(yǎng)基中生長1h，DCFHDA染色30 min。熒光顯微鏡下拍照，A：15 min，B：30 min，C：60 min。熒光酶標儀所測熒光強度(D)，所有值均為三次平行實驗的平均值177。標準誤（n=3）。使用SPSS進行T檢驗，與CK相比，t ：差異顯著(*)，t ：差異極顯著(**) 。與10%E相比，t ：差異顯著()，t ：差異極顯著()。. Effects of GSH、Met and NAC on the H2O2 level of cells under ethanol stress. Cell were grown in YPD medium until midlog phase and treated with indicated reagents for 1 h. Cells were stained with DCFHDA for 30 min, fluorescent micrographs of the cells were taken under a fluorescence microscope. The cells were treated for 15 min (A), 30 min (B), 60 min (C). The relative fluorescence intensity were measured by a microplate reader(D). CK: control, 10%E: 10% ethanol, 10%E+GSH: 10% ethanol and mM GSH, 10%E+Met: 10% ethanol and 6 mM Met, 10%E+NAC: 10% ethanol and 10mM NAC. Each rectangle represents the mean 177。SE of three replicates (n=3). Compared with CK, differences were significant at t (*) andt (**) and pared with 10%E, differences were significant at t () and t (), according to ttest.H2O2本身幾乎沒有活性，但是可以自由的穿過細胞膜轉(zhuǎn)化成強氧化性的羥自由基。為了檢測H2O2濃度，將處理過的細胞用DCFHDA探針孵育30 min，分別采用熒光顯微鏡和熒光酶標儀拍攝圖片和測定熒光強度。熒光染料DCFHDA（2’，7’二氫二氯熒光黃雙乙酸鈉）可以自由穿過細胞膜進入細胞內(nèi)，在胞內(nèi)非特異性脂酶的催化下，變成DCFH (2’，7’二氫二氯熒光黃)，DCFHDA本身并不能發(fā)出熒光。當細胞內(nèi)有H2O2存在時，DCFHDA就會被氧化成DCF (2’，7’二氯熒光黃)，在激光激發(fā)下發(fā)出綠色熒光，波長在524 nm左右。其熒光強度可反映H2O2的水平。，與對照相比，用10%乙醇處理15 min就會導致其熒光強度的增加，說明釀酒酵母細胞內(nèi)H2O2水平與乙醇處理時間呈正相關(guān)。在乙醇存在時分別加GSH，Met和NAC的三組處理在15 min，30 min時熒光強度與單獨的乙醇處理組沒有明顯差別，而在60 min時三者明顯降低了熒光強度。結(jié)果表明GSH, Met和NAC可以恢復乙醇增加的H2O2含量，但是是在后期起作用（60 min）。釀酒酵母在含有10%乙醇的YPD液體培養(yǎng)基中培養(yǎng)60 min，以細胞中MDA含量的高低代表其細胞膜脂過氧化水平。結(jié)果顯示，乙醇脅迫下酵母細胞MDA含量顯著升高，幾乎為對照的三倍，而GSH、Met和NAC可以顯著地降低乙醇導致的MDA水平上升，說明GSH、Met和NAC可以恢復乙醇導致的膜脂過氧化水平（）。 GSH，Met和NAC對乙醇脅迫釀酒酵母MDA含量的影響。培養(yǎng)至對數(shù)期的酵母分別在含H2O、10% ethanol、10% ethanol 和 mM GSH、10% ethanol和 6 mM Met、10% ethanol 和10 mM NAC的YPD培養(yǎng)基中生長1 h ，硫代巴比妥法測定MDA含量。所有值均為三次平行實驗的平均值177。標準誤（n=3）。使用SPSS進行T檢驗，與CK相比，t ：差異顯著(*)，P ：差異極顯著(**) 。與10% E相比，t ：差異顯著()，t ：差異極顯著()。. Effects of GSH、Met and NAC on the MDA concent of cells under ethanol stress. Cell were grown in YPD medium until midlog phase and treated with indicated reagents for 1 h. CK: control, 10% E: 10% ethanol, 10% E+GSH: 10% ethanol and mM GSH, 10% E+Met: 10% ethanol and 6 mM Met, 10% E+NAC: 10% ethanol and 10 mM NAC. Each rectangle represents the mean 177。SE of three replicates (n=3). Compared with CK, differences were significant at t (*) and t (**) and pared with 10%E, differences were significant at t () and t (), according to ttest. GSH、Met和NAC對乙醇產(chǎn)率的影響 GSH，Met和NAC對乙醇脅迫釀酒酵母乙醇產(chǎn)率的影響。培養(yǎng)至對數(shù)期的酵母分別在含H2O、 mM GSH、6 mM Met、10 mM NAC的發(fā)酵培養(yǎng)基中生長72 h，蒸餾后采用密度瓶法檢測酒精濃度。所有值均為三次平行實驗的平均值177。標準誤（n=3）。使用SPSS進行T檢驗，與CK相比，t ：差異顯著(*)，t ：差異極顯著(**) 。. Effects of GSH、Met and NAC on the ethanol yield of cells under ethanol stress. Cell were grown in fermentation medium until midlog phase and treated with indicated reagents for 72 h.