【正文】 使用SPSS進(jìn)行T檢驗(yàn)，與CK相比，t ：差異顯著(*)，t ：差異極顯著(**) 。所有值均為三次平行實(shí)驗(yàn)的平均值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。與10% E相比，t ：差異顯著()，t ：差異極顯著()。標(biāo)準(zhǔn)誤（n=3）。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。結(jié)果顯示，乙醇脅迫下酵母細(xì)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(dǎo)致的MDA水平上升，說明GSH、Met和NAC可以恢復(fù)乙醇導(dǎo)致的膜脂過氧化水平（）。結(jié)果表明GSH, Met和NAC可以恢復(fù)乙醇增加的H2O2含量，但是是在后期起作用（60 min）。與對照相比，用10%乙醇處理15 min就會導(dǎo)致其熒光強(qiáng)度的增加，說明釀酒酵母細(xì)胞內(nèi)H2O2水平與乙醇處理時(shí)間呈正相關(guān)。當(dāng)細(xì)胞內(nèi)有H2O2存在時(shí)，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。為了檢測H2O2濃度，將處理過的細(xì)胞用DCFHDA探針孵育30 min，分別采用熒光顯微鏡和熒光酶標(biāo)儀拍攝圖片和測定熒光強(qiáng)度。. 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。使用SPSS進(jìn)行T檢驗(yàn)，與CK相比，t ：差異顯著(*)，t ：差異極顯著(**) 。熒光酶標(biāo)儀所測熒光強(qiáng)度(D)，所有值均為三次平行實(shí)驗(yàn)的平均值177。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。的敏感性不同造成的。含量。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產(chǎn)生的多少。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與10%E相比，t ：差異顯著()，t ：差異極顯著()。標(biāo)準(zhǔn)誤（n=3）。酶標(biāo)儀所測吸光度值(B)。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。含量。以上結(jié)果表明乙醇處理增加了細(xì)胞內(nèi)O2雖然活性不強(qiáng)，但可以直接與某些蛋白發(fā)生反應(yīng)，造成蛋白損傷；與羥基（OH）結(jié)合后的產(chǎn)物還會導(dǎo)致細(xì)胞DNA損壞，破壞細(xì)胞機(jī)體功能。. 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。標(biāo)準(zhǔn)誤（n=3）。熒光酶標(biāo)儀所測熒光強(qiáng)度(B)。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。和H2O2的水平。、H2O2等。. Effects of GSH、Met and NAC on the Mitochondrial Membrane Potential of cells under ethanol stress. Cell were grown in YPD medium until midlog phase and treated with indicated reagents for given times. 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. Cells were stained with Rh123 for 30 min, fluorescent micrographs of the cells were taken under a fluorescence microscope. 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。使用SPSS進(jìn)行T檢驗(yàn)，與CK相比，P ：差異顯著(*)，P ：差異極顯著(**) 。熒光顯微鏡下拍照（A），熒光酶標(biāo)儀所測熒光強(qiáng)度(B)，所有值均為三次平行實(shí)驗(yàn)的平均值177。 GSH、Met和NAC對乙醇脅迫釀酒酵母線粒體膜電位的影響。在此基礎(chǔ)上，采用Rh123檢測釀酒酵母線粒體膜電位的變化。羅丹明123是一種可透過細(xì)胞膜的陽離子熒光染料，是一種線粒體跨膜電位的指示劑。分別處理30 min時(shí)，三者都出現(xiàn)了紅色熒光不同程度降低的現(xiàn)象，其中GSH和NAC極其顯著地降低了由乙醇引起的熒光強(qiáng)度增加，各組處理1 h后結(jié)果發(fā)現(xiàn)，與乙醇處理的酵母細(xì)胞相比，GSH、Met和NAC三者都極其顯著地降低了熒光強(qiáng)度。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. ，與對照相比，10%乙醇處理15 min后的釀酒酵母細(xì)胞的紅色熒光即有明顯增強(qiáng)，隨著處理時(shí)間的延長，其增加更為顯著。與10% E相比，P ：差異顯著()，P ：差異極顯著()。標(biāo)準(zhǔn)誤（n=3）。熒光顯微鏡下拍照，A：15 min，B：30 min，C：60 min。 GSH、Met和NAC對乙醇脅迫的釀酒酵母細(xì)胞膜完整性的影響。通過PI染色檢驗(yàn)GSH，Met和NAC對乙醇脅迫釀酒酵母細(xì)胞膜完整性的恢復(fù)作用。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. 細(xì)胞膜是一道防止細(xì)胞外物質(zhì)自由進(jìn)入細(xì)胞的天然屏障，主要功能是保證細(xì)胞內(nèi)環(huán)境的相對穩(wěn)定，從而使各種生化反應(yīng)能夠有序良好運(yùn)行。與10%E相比，P ：差異顯著()，P ：差異極顯著()。標(biāo)準(zhǔn)誤（n=3）。C下孵育72 h，記菌落數(shù)。B)平板梯度稀釋法測定細(xì)胞存活率。A) 取經(jīng)過不同化合物處理1h后的對數(shù)期釀酒酵母，按10倍梯度稀釋過后，各取2μl點(diǎn)在YPD固體培養(yǎng)基上，將點(diǎn)有不同濃度梯度酵母細(xì)胞的YPD固體平板于30176。結(jié)果表明GSH，Met 和 NAC對乙醇脅迫的酵母可能具有恢復(fù)作用。為了探明這三種化合物是否在乙醇脅迫中起作用，分別將GSH，Met和NAC加入含有10%乙醇的YPD培養(yǎng)基中，處理1 h。SAM是細(xì)胞正常功能和生存的重要分子之一，是最重要的甲基供體，作為使用最廣泛的輔助因子僅次于ATP[36]。NAC是細(xì)胞內(nèi)GSH的還原型谷胱甘肽的前體，能快速穿過細(xì)胞膜，是一種生物學(xué)研究中廣泛使用的抗氧化劑。GSH不僅可以清除活性氧，而且可以調(diào)節(jié)基因表達(dá)，氧化還原信號和酶活性等[35]。結(jié)果表明乙醇可以明顯降低膜的完整性，且具有濃度依賴性。釀酒酵母細(xì)胞分別用不同濃度乙醇處理1 h之后，進(jìn)行PI染色發(fā)現(xiàn)，釀酒酵母細(xì)胞膜完整性與添加的乙醇濃度呈負(fù)相關(guān)，與對照相比，隨著乙醇濃度的增加，PI染色的細(xì)胞發(fā)出的紅色熒光也隨著增強(qiáng)，呈濃度依賴性?；谶@個(gè)原因，PI被廣泛用于細(xì)胞膜完整性的檢測。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. 為了探明乙醇是否對細(xì)胞膜造成損傷，本實(shí)驗(yàn)采用熒光酶標(biāo)儀檢測了碘化丙啶（Propidium, PI）染色細(xì)胞樣品的熒光強(qiáng)度。與CK相比，P ：差異顯著(*)，P ：差異極顯著(**)，與10%E相比，P ：差異顯著()，P ：差異極顯著()。所有值均為三次平行實(shí)驗(yàn)的平均值177。 所示，處理時(shí)間為15 min時(shí)，5%無水乙醇已對酵母細(xì)胞的生長有輕微的抑制作用，而10%的乙醇則有明顯的抑制作用，隨著時(shí)間的延長，5%和10%兩種處理對酵母生長的抑制作用依然非常明顯()。C下孵育72 h后拍照。 不同濃度乙醇對釀酒酵母菌落生長的影響。SE of three replicates (n=3). 活化后生長至對數(shù)期的釀酒酵母菌株分別在含有0，5 %和10%無水乙醇的YPD液體培養(yǎng)基中培養(yǎng)3 h，隔1小時(shí)取一次樣，采用平板計(jì)數(shù)法測定菌落數(shù)。Fig. . Effects of differnent concentration of ethanol on the viability of cells. Cell were grown in YPD meidium until midlog phase and treated with water, 5% ethanol or 10% ethanol. Cell viability was determined by standard dilution plate counts on solid YPD medium. The plates were incubated at 30176