【作者】 刘晶；

【导师】 张英春；

【作者基本信息】 哈尔滨工业大学 ， 化学工程， 2019， 硕士

【摘要】 S-层蛋白是乳杆菌表面结构的主要成分,占细胞总蛋白的10%-15%,占网格表面的70%。据报道S-层蛋白对乳杆菌益生菌的作用有积极的贡献。目前关于S-层蛋白对宿主细胞的线粒体凋亡通路的影响的研究较少,所以本课题以H₂O₂作为氧化损伤刺激物,研究从Lactobacillus paracasei subsp.paracasei M5-L、Lactobacillus casei Q8-L及Lactobacillus rhamnosus GG（LGG）三种乳酸杆菌上分离的S-层蛋白对HT-29细胞的线粒体凋亡通路的影响。本研究采取5 mol/L的LiCl提取S-层蛋白,并利用1 mol/L的LiCl通过盐析的方法来纯化粗提蛋白,SDS-PAGE检测表明三株菌均在43 kDa处有单一条带。选择不同浓度S-层蛋白作用细胞,经MTT实验最终确定其作用浓度为150μg/mL,活菌体与热灭活菌体的浓度为10⁷ cfu/mL和10⁸ cfu/mL,H₂O₂与细胞作用的最佳浓度是0.8 mM,作用时间为6 h进行后续实验。MTT实验证实作用时间为6 h时,三种S-层蛋白、活菌体与热灭活菌体能够抑制H₂O₂对HT-29细胞增殖的影响。检测HT-29细胞抗氧化酶系统的T-AOC、SOD、CAT、MDA和LDH五个指标,实验证明三种S-层蛋白可以通过提高T-AOC、SOD和CAT的含量,降低MDA和LDH的含量,进而拮抗H₂O₂对HT-29细胞的影响。三种S-层蛋白的各处理组对H₂O₂导致的Caspase-3和Caspase-9活性的异常具有显著的正向调节作用。实验结果表明,三种S-层蛋白均能通过降低其的活化作用进而减缓H₂O₂引起的HT-29的凋亡。通过流式细胞仪对三种S-层蛋白及H₂O₂对HT-29细胞中线粒体膜电位的变化及对细胞中Ca²⁺的影响两个凋亡指标的研究,进而确定M5-L的S-层蛋白（预防组）拮抗H₂O₂对HT-29的作用效果最佳,所以最终选择M5-L的S-层蛋白（预防组）来进行细胞凋亡机制部分的研究。为探讨三种S-层蛋白拮抗H₂O₂的机制,利用qRT-PCR检测HT-29细胞线粒体凋亡通路相关mRNA表达情况,实验结果表明:S-层蛋白可以通过降低APAF-1、Caspase-3、Caspase-9、CytC、Smac和Bid基因的表达量,升高XIAP和Bcl-2基因的表达量,进而通过线粒体细胞凋亡通路来抑制H₂O₂诱导的细胞凋亡作用。由于AIFM1和ENDOG基因相对其他基因表达含量过少,从本实验的结果看各组的AIFM1和ENDOG的变化趋势不是很有规律,是否M5-L的S-层蛋白通过非Caspase依赖的AIFM1和ENDOG途径抑制细胞调亡仍有待进一步研究。更多还原

【Abstract】 The S-layer protein is the main component of the surface structure of lactobacillus,accounting for 10%-15%of the total protein of the cell and 70%of the surface of the grid.It has been reported that the S-layer protein has a positive contribution to the probiotics of lactobacillus.Currently,there are few studies on the effect of S-layer protein on the mitochondrial apoptosis pathway of host cells.Therefore,in this study,H₂O₂ was used as an oxidative damage stimulant to study the effects of S-layer proteins isolated from Lactobacillus paracasei subsp.paracasei M5-L,Lactobacillus casei Q8-L and Lactobacillus rhamnosus GG（LGG）on the mitochondrial apoptosis pathway of HT-29 cells.In this study,5 mol/l LiCl was used to extract S-layer protein,and 1 mol/l LiCl was used to purify crude extracted protein by salting out.SDS-PAGE test showed that both strains had a single band at 43 kDa.After adjusting the concentration of S-layer protein,the concentration of active and heat-inactivated bacteria was finally determined to be 150μg/ml by MTT experiment,the concentration of live and heat-inactivated bacteria was 10⁷ cfu/ml and 10⁸ cfu/ml,and the optimal concentration of H₂O₂ to interact with cells was 0.8 mM,and the action time was 6 h for follow-up experiments.MTT experiment confirmed that three S-layer proteins,live bacteria and heat-inactivated bacteria could inhibit the effect of H₂O₂ on HT-29cell proliferation at the action time of 6 h.The five indexes of T-AOC,SOD,CAT,MDA and LDH of the antioxidant enzyme system in HT-29 cells were detected.The experiments showed that the S-layer proteins of the three strains could increase the content of T-AOC,SOD and CAT and reduce the content of MDA and LDH,thereby antagonizing the effect of H₂O₂ on HT-29 cells.Each treatment group of the three S-layer proteins had a significant positive regulatory effect on the abnormal activity of caspase-3 and caspase-9 caused by H₂O₂.The results showed that the S-layer proteins of M5-L,Q8-L and LGG could slow down H₂O₂-induced HT-29 apoptosis by reducing their activation.By flow cytometry of three strains of S-layer of proteins and H₂O₂ in the cells of HT-29 in the change of mitochondrial membrane potential and cellular Ca²⁺two apoptosis index of the study,the influence of layer,in turn,determined the M5-L S-layer protein group（prevention）antagonism H₂O₂ for HT-29 cell function,the optimal,so eventually chose the M5-L S-layer protein group（prevention）for studying the mechanism of apoptosis part.To improve the mechanism of three S-layer proteins antagonizing H₂O₂,real-time fluorescence quantitative PCR was used to detect the expression of mRNA related to mitochondrial apoptosis pathway in HT-29 cells.The results showed that the S-layer protein of lactobacillus can increase the expression of XIAP and Bcl-2genes by reducing the expression levels of APAF-1,Caspase-3,Caspase-9,CytC,Smac and Bid genes,and then inhibited the apoptosis induced by H₂O₂ through the mitochondrial cell apoptosis pathway.Due to the low expression content of AIFM1and ENDOG genes compared with other genes,the change trend of AIFM1 and ENDOG in each group was not very regular according to the results of this experiment.Whether the S-layer protein of M5-L inhibited cell apoptosis through AIFM1 and ENDOG pathways that were not caspase-dependent remained to be further studied.更多还原