【摘要】：本論文對酵母細(xì)胞在高滲、低溫和氧化條件下的應(yīng)激反應(yīng)進(jìn)行了研究,篩選了耐高滲酵母突變株,通過低劑量刺激酵母細(xì)胞產(chǎn)生活性衍生物—LYCD,開辟了制備LYCD的新途徑,考察了LYCD對氧化損傷的酵母細(xì)胞的保護(hù)作用。 采用對數(shù)期正常酵母細(xì)胞為研究對象,對其進(jìn)行低劑量高滲(KCl)和氧化(H_2O_2)預(yù)處理后發(fā)現(xiàn)細(xì)胞對更高劑量產(chǎn)生了抗性,并且這種抗性具有自我適應(yīng)性和交叉適應(yīng)性。此外,在低溫和氧化應(yīng)激反應(yīng)之間也存在交叉適應(yīng)性。這種交叉適應(yīng)性說明,高滲和氧化應(yīng)激、低溫和氧化應(yīng)激對細(xì)胞的損傷在一定程度上具有相似性,預(yù)處理后可能誘導(dǎo)出相似的保護(hù)物質(zhì)。 測定了酵母經(jīng)高滲、低溫和氧化預(yù)處理前后細(xì)胞內(nèi)幾種重要抗氧化劑(GSH、SOD、CAT)和總抗氧化能力(T-AOC)的變化,結(jié)果表明:預(yù)處理都可以使正常酵母細(xì)胞中抗氧化劑含量提高,總抗氧化能力增強(qiáng)。并分析了正常酵母預(yù)處理前后細(xì)胞內(nèi)脂質(zhì)過氧化物—丙二醛(MDA)的含量,發(fā)現(xiàn)預(yù)處理后,再置于氧化致死條件下,MDA的含量降低,細(xì)胞的存活率也升高。這不僅說明預(yù)處理使細(xì)胞抗氧化水平提高,減少了MDA的積累,細(xì)胞增加了對這幾種不良環(huán)境的抗性,更直接說明高滲、低溫與氧化關(guān)系密切。 將經(jīng)過高滲和低溫預(yù)處理后的酵母進(jìn)行破壁,分別得到LYCD_(KCl)和LYCD_(LT),發(fā)現(xiàn)兩者都對細(xì)胞抗氧化具有保護(hù)活性。分別對其處理?xiàng)l件進(jìn)行優(yōu)化,確定了LYCDK_(KCl)的最佳處理?xiàng)l件為:1%KCl預(yù)處理90min,LYCD_(LT)的最佳處理?xiàng)l件為:4℃預(yù)處理60min。 通過紫外誘變篩選了耐高滲酵母突變株(CYK),與正常酵母相比,發(fā)現(xiàn)耐高滲突變株對氧化具有很強(qiáng)的抗性,而且耐高滲酵母提取物(LYCDK)也具有保護(hù)細(xì)胞抵抗氧化的能力。分析了正常酵母和耐高滲酵母以及預(yù)處理對耐高滲酵母細(xì)胞內(nèi)GSH、SOD、CAT、T-AOC和MDA的差異,發(fā)現(xiàn)耐高滲酵母胞內(nèi)抗氧化水平高于正常酵母菌株,且預(yù)處理增加了LYCDK中抗氧化劑的含量,降低了MDA水平。 對正常酵母細(xì)胞預(yù)處理前后蛋白的變化進(jìn)行了電泳分析,結(jié)果表明,一些蛋白是H_2O_2和KCl都可誘導(dǎo)合成的,另一些蛋白是由H_2O_2或KCl特異性誘導(dǎo)合成的。 探討了H_2O_2和紫外線條件下,LYCD對酵母細(xì)胞氧化損傷的保護(hù)作用。在H_2O_2(10mmol/L)和紫外線(20W紫外燈下30cm處照射60s)氧化損傷酵母細(xì)胞模型中添加LYCD后,細(xì)胞存活率顯著上升,且有劑量依賴性。LYCD提高了紫外線氧化損傷細(xì)胞上清液中GSH、SOD和CAT含量,降低了MDA水平。
[Abstract]:In this paper, the stress reaction of yeast cells under hyperosmotic, low temperature and oxidizing conditions was studied. The mutant strain of hyperosmotic yeast was screened, and a new way to prepare LYCD was opened up by low dose stimulation of yeast cells to produce active derivative-LYCD,. The protective effect of LYCD on oxidative damaged yeast cells was investigated. Normal yeast cells in logarithmic phase were pretreated with low dose and high osmotic (KCl) and oxidation (H_2O_2). It was found that the cells developed resistance to higher doses and the resistance was self-adaptive and cross-adaptive. In addition, there is cross adaptation between low temperature and oxidative stress reaction. This cross-adaptation indicated that the damage to cells caused by hyperosmotic and oxidative stress, hypothermia and oxidative stress was similar to a certain degree, and similar protective substances might be induced after pretreatment. The changes of several important antioxidants (GSH,SOD,CAT) and total antioxidant capacity (T-AOC) in yeast before and after hypertonic, low temperature and oxidation pretreatment were measured. The results showed that pretreatment could increase the content of antioxidants in normal yeast cells. Total antioxidant capacity increased. The content of lipid peroxide-malondialdehyde (MDA) in normal yeast cells was analyzed before and after pretreatment. It was found that after pretreatment, the content of MDA decreased and the survival rate of cells increased. This indicated that pretreatment not only increased the antioxidant level of cells and reduced the accumulation of MDA, but also increased the resistance of cells to these adverse environments, which directly indicated that high permeability and low temperature were closely related to oxidation. The yeast treated with hypertonic and cryogenic pretreatment was broken into the wall, and LYCD_ (KCl) and LYCD_ (LT), were obtained, respectively, which showed that both of them had protective effect on the antioxidant activity of the cells. The optimum treatment conditions of LYCDK_ (KCl) were determined as follows: 1%KCl pretreatment 90 min LYCD _ (LT) treatment conditions were: 4 鈩，

本文編號：2272359