S.boulardii预防DON诱导猪巨噬细胞凋亡的作用机理研究

发布时间：2018-05-22 15:32

本文选题：呕吐毒素 + 布拉迪酵母　；参考：《武汉轻工大学》2015年硕士论文

【摘要】：真菌毒素在粮食生产及饲料行业中的污染一直备受关注,单端孢霉烯族毒素的临床症状有腹泻、呕吐、出血,严重时会导致死亡性休克,危害人畜健康。脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON),又称呕吐毒素,由镰刀菌属、葡萄穗霉属等真菌产生。虽然毒性较低,但因其稳定性高,并且在农作物如小麦、燕麦和大麦中广泛存在,从而得到更广泛的研究。国内外学者在DON诱导的免疫细胞毒性方面取得了一定的进展,但对于如何干预并降低这一毒素引发的慢性中毒仍然亟待解决。益生菌作为一种新型的免疫调节剂,在改善肠道营养,调节机体免疫方面发挥着重要作用。布拉迪酵母(Saccharomyces c e re v i s i a e b o u l a rd i i,S.b o u l a rd i i)是唯一一种具有益生作用的酵母菌,对于生物毒素引发的腹泻有着良好的疗效。对于布拉迪酵母是否具有对DON诱导的免疫细胞凋亡的干预作用以及可能存在的作用分子机理,并未见有文献报道。本研究旨在通过以猪肺泡巨噬细胞(Porcine Alveolar Macrophage,PAM)为免疫细胞模式株,将DON作用于PAM,诱导其产生细胞凋亡,在此细胞凋亡模型基础上,探究不同浓度的S.boulardii对DON损伤细胞的干预作用,并以两者作用的共同通路—p38MAPK通路为靶点,研究不同剂量S.boulardii对p38MAPK通路的下游炎性因子分泌以及p38蛋白表达的影响,进而阐明S.boulardii干预作用可能的分子机理。再者为进一步探究酵母菌中是何种成分在发挥作用,因细胞壁为酵母菌与外界接触的直接感应点,本研究初步探讨了S.boulardii细胞壁对损伤细胞炎性因子m RNA表达量的影响。主要研究结果如下:1.通过M TT实验与A nnex in-V F ITC/P I双染法检测细胞凋亡的结果显示,DON毒素对PAM的毒性作用呈现时间和剂量依赖性,随毒素浓度升高,对细胞生长的抑制率逐渐增大,凋亡率上升。利用ELISA等手段检测DON对炎性因子IL-6、TNF-α和IL-lβ分泌量的影响结果显示,在DON浓度为4μM时,炎性因子随着DON的作用时间呈先升后降的趋势,4h时显著升高。炎性因子m R NA表达量随DO N浓度升高呈上升趋势,8μM时呈极显著差异(P0.01)。因此,确定诱导细胞凋亡模型的DON毒素作用时间为4h,浓度为8μM,用于后续酵母菌干预效果研究。2.在DON对S.boulardi i未造成毒性损伤的前提下,酵母菌与DON共同作用于PAM时,采用流式细胞仪检测细胞凋亡的结果显示:随布拉迪酵母浓度升高,降低了PAM的凋亡率,同时抑制了DON对炎性因子IL-6、TNF-α和IL-lβ表达量的上调,较高浓度S.boulardii(5×106个/m L)作用显著(P0.05)。对于凋亡调控基因Bcl-2/Bax与Bcl-xl/Bax,中剂量的S.boulardii相比DON毒素处理组极显著(P0.01)上调了该比值,促使细胞发挥抗凋亡作用。DON明显上调了p38的m RNA表达,中剂量(5×105个/m L)的S.boulardii则极显著(P0.01)下调了p38m RNA表达,与抑制剂SB203580同样起到抑制作用。West ernbl ot检测p3 8蛋白组表达水平结果显示,S.boulardi i能够抑制p 3 8蛋白的磷酸化,阻滞p38MAPK通路的激活。3.随酵母菌细胞壁浓度升高,各炎性因子的m RNA表达量均逐渐升高,炎性因子IL-6和TNF-α的中高剂量组(10mg/L和20mg/L)均高于DON处理组的表达量,各酵母细胞壁组仅对因子IL-1β有一定抑制作用。以上结果表明,S.boulardii能够降低p38的表达,并且抑制DON对p38 M AP K通路的激活而降低下游炎性因子的表达,同时上调抗凋亡因子的表达,从而抑制细胞凋亡。S.boulardii细胞壁在一定程度上激活了细胞的免疫反应,S.boulardii发挥抑制作用的活性成分仍需进一步探究。
[Abstract]:The contamination of mycotoxins in the food production and feed industry has been paid much attention. The clinical symptoms of the monosamycin are diarrhea, vomiting, and bleeding, which can lead to death shock and harm to human and animal health. Deoxynivalenol (deoxynivalenol, DON), also known as vomit toxin, is produced by fungi from the genus Fusarium and grape panicle. Although low toxicity, but because of its high stability and widespread in crops such as wheat, oat and barley, more extensive studies have been made. Scholars both at home and abroad have made some progress in DON induced immune cytotoxicity, but it is still urgent to solve the problem of how to intervene and reduce the chronic poisoning caused by this toxin. As a new immunomodulator, it plays an important role in improving intestinal nutrition and regulating body immunity. Saccharomyces c e re v i s I a e b o has a good effect on diarrhoea caused by biological toxin. No literature has been reported on whether Brady's yeast can interfere with DON induced apoptosis and the possible molecular mechanism. The aim of this study is to induce DON to induce apoptosis by using DON Alveolar Macrophage (PAM) as an immune cell model strain, and to induce DON to act on PAM. On the basis of this cell apoptosis model, the effects of different concentrations of S.boulardii on DON damaged cells were explored, and the effects of different doses of S.boulardii on the secretion of inflammatory factors and the expression of p38 protein in the downstream of p38MAPK pathway were studied by the common pathway of the two actions, and the effect of S.boulardii intervention was clarified. The molecular mechanism of energy is further explored to further explore the role of the yeast in the yeast, because the cell wall is the direct contact between the yeast and the outside world. The effect of S.boulardii cell wall on the expression of M RNA in damaged cells is preliminarily investigated. The main results are as follows: 1. through the M TT experiment and A nnex in-V F The results of cell apoptosis detected by ITC/P I double staining showed that the toxic effect of DON toxin on PAM showed time and dose dependence. With the increase of the concentration of toxin, the inhibition rate of cell growth increased gradually and the rate of apoptosis increased. The effects of DON on the secretion of IL-6, TNF- A and IL-l beta were detected by ELISA and so on, and the concentration of DON was shown to be in DON concentration. At 4 M, the inflammatory factors rose and then decreased with the time of the action of DON. The expression of the inflammatory factor M R NA increased with the increase of the concentration of DO N (P0.01). Therefore, the DON toxin action time of the induced apoptosis model was identified as 4h, the concentration was 8 micron, and was used for the follow-up effect of yeast. Under the premise that DON did not cause toxic damage to S.boulardi I, the results of cell apoptosis detected by the flow cytometer by yeast and DON showed that with the increase of Brady's yeast concentration, the apoptosis rate of PAM was reduced and the expression of DON against inflammatory factors IL-6, TNF- A and IL-l beta was up-regulated, and a higher concentration of S.boul was inhibited by the.2.. The effect of ardii (5 x 106 /m L) was significant (P0.05). As for the apoptosis regulation gene Bcl-2/Bax and Bcl-xl/Bax, the median dose of S.boulardii was significantly higher than that in the DON toxin treatment group (P0.01), which promoted the cell to play the anti apoptosis role of.DON obviously up the p38 M expression, and the median dose (5 x 105) was extremely significant. The expression of p38m RNA was adjusted, and the inhibitory effect of inhibitor SB203580 on the expression of.West ernbl ot in P3 8 protein group showed that S.boulardi I could inhibit the phosphorylation of P 38 protein, and the activation.3. of p38MAPK pathway increased with the increase of yeast cell wall concentration, and the expression of every inflammatory factor increased gradually. The middle and high dose group of 6 and TNF- a (10mg/L and 20mg/L) were higher than that of DON treatment group, and the cell wall group of each yeast had a certain inhibitory effect on the factor IL-1 beta. The above results showed that S.boulardii could reduce the expression of p38 and inhibit the activation of p38 M AP K pathway to lower the expression of downstream inflammatory factors and up regulate the anti apoptotic factors. The expression of the subcell, which inhibits the apoptosis of.S.boulardii cell wall, activates the cell's immune response to a certain extent, and the active components of the inhibitory action of S.boulardii still need to be further explored.
【学位授予单位】：武汉轻工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.8

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