四株乳酸杆菌对溃疡性肠道炎症的调节作用研究

发布时间：2018-06-11 23:46

本文选题：乳酸杆菌 + 肠道炎症　；参考：《哈尔滨工业大学》2017年硕士论文

【摘要】：乳酸杆菌属于肠道正常菌群,参与调节机体免疫应答,以维持肠道微生态的平衡。越来越多的研究发现,乳酸杆菌作为一种益生菌,具有多种免疫调节活性。体内和体外实验都证实乳酸杆菌能够调节肠炎。但是,目前关于乳酸杆菌对肠炎的调节作用同时进行体内和体外验证研究较少。因此本课题以脂多糖(LPS)作为炎性刺激物,研究从L.paracasei subsp.paracasei M5-L、L.casei Q8-L、L.rhamnosus J10-L和LGG对HT-29细胞炎性因子调节作用,并为动物实验筛选出对肠炎抑制效果更为显著的乳杆菌。随后进行动物实验验证,动物实验中以葡聚糖硫酸钠(DSS)作为炎性刺激物,建立溃疡性肠炎模型,研究乳杆菌对小鼠溃疡性肠道炎症的调节作用。体外实验首先对四株乳杆菌J10-L、Q8-L、M5-L和LGG进行计数,利用MTT实验确定后续实验条件为LPS作用浓度为5μg/m L,乳酸杆菌与细胞的感染复数为1:1,同时证实了选定的四株乳杆菌都能够抑制LPS对HT-29细胞增殖的影响,但是不同菌株在不同时间对细胞增殖的影响不同。通过NO试剂盒测定细胞上清液中NO,ELISA试剂盒测定细胞上清液中IL-8和TNF-α含量的检测,证明四株乳酸杆菌都能够明显抑制LPS刺激细胞过度分泌NO和TNF-α,并且这种抑制作用存在菌株差异性。但是对IL-8的下调作用并不显著。体外细胞实验结果表明乳杆菌J10-L对肠炎的调节作用效果不显著,并且四株菌株预防组效果相对于治疗组和竞争组效果更显著。根据体外细胞实验结果筛选出对肠道炎症因子抑制效果更显著的乳酸杆菌Q8-L、M5-L和LGG进行小鼠肠炎预防实验。体内小鼠实验通过预实验确定用2%DSS来诱导肠炎模型,并在诱导小鼠肠炎模型前用乳杆菌浓度为108 cfu/m L和109 cfu/m L的乳杆菌Q8-L、M5-L和LGG灌胃4周。养鼠期间每天测定小鼠的体重并根据体重减轻情况和血便情况对小鼠进行DAI评分,DAI评分表明三株乳杆菌能不同程度的减轻小鼠患病症状。随后通过小鼠脏器重量和结肠长度表明三株乳酸杆菌均能肠炎引起的脾重量增加和结肠变短变轻状况。对小鼠结肠进行HE染色后,观察组织切片并且进行组织学评分证实Q8-L、M5-L和LGG能不同程度的缓解肠炎带来的组织学损伤状况。通过MPO试剂盒测定小鼠结肠中的MPO,ELISA法测定小鼠血清中IL-8和TNF-α含量,发现乳酸杆菌Q8-L、M5-L和LGG都能下调DSS刺激的小鼠产生的MPO,IL-8和TNF-α含量,上调IL-10含量。用RT-PCR测定TLR4和TNF-α的基因表达量,在基因水平上说明三株乳酸杆菌能抑制DSS刺激小鼠分泌的TNF-α,并且TLR4受体在乳杆菌调节肠道炎症时起到关键作用。这些结果表明乳杆菌能够显著调节体内肠炎,并且调节作用存在菌株和浓度差异性。
[Abstract]:Lactobacillus belongs to the normal intestinal flora and participates in the regulation of immune response in order to maintain the balance of intestinal microecology. More and more studies have found that Lactobacillus, as a probiotic, has a variety of immunomodulatory activities. In vivo and in vitro experiments have shown that Lactobacillus can regulate enteritis. However, there are few studies on the regulatory effect of Lactobacillus on enteritis in vivo and in vitro. In this study, lipopolysaccharide (LPS) was used as an inflammatory stimulant to study the regulatory effects of L.paracasei subsp.paracasei M5-L5 Lcasei Q 8-L Lrhamnosus J10-L and LGG on the inflammatory cytokines of HT-29 cells, and to screen out Lactobacillus with more significant inhibitory effect on enteritis in animal experiments. Then the animal experiment was carried out. In the animal experiment, sodium dextran sulfate (DSS) was used as the inflammatory stimulant to establish the model of ulcerative enteritis, and to study the regulatory effect of Lactobacillus on ulcerative intestinal inflammation in mice. In vitro, four strains of Lactobacillus, J10-L, Q8-L, M5-L and LGG, were counted. MTT assay was used to determine the following experimental conditions: LPS concentration was 5 渭 g / mL, and the number of infection between Lactobacillus and cells was 1: 1. It was also confirmed that all four strains of Lactobacillus could inhibit the proliferation of HT-29 cells induced by LPS. But different strains have different effects on cell proliferation at different time. The content of IL-8 and TNF- 伪 in the supernatant was determined by no Elisa kit. The results showed that all four strains of Lactobacillus could inhibit the excessive secretion of no and TNF- 伪 by LPS-stimulated cells, and the inhibition was different. But the down-regulation of IL-8 was not significant. The results of cell experiment in vitro showed that the regulatory effect of Lactobacillus J10-L on enteritis was not significant, and the effect of four strains in prevention group was more significant than that in treatment group and competitive group. According to the results of cell experiment in vitro, we selected Lactobacillus Q8-LM5-L and LGG to prevent enteritis in mice. The mouse model of enteritis was induced by 2DSS in vivo. Before inducing enteritis in mice, Lactobacillus Q8-LnM5-L and LGG were perfused with Lactobacillus Q8-LM5-L and LGG for 4 weeks before inducing the model of enteritis with Lactobacillus at concentrations of 108 cfu/m / L and 109 cfu/m / L. The body weight of the mice was measured every day during the incubation period and the Dai score showed that the three strains of Lactobacillus could alleviate the symptoms of the mice in varying degrees according to the weight loss and blood stool. Then the weight of viscera and the length of colon in mice showed that all three strains of Lactobacillus could increase spleen weight and shorten colon induced by enteritis. After colonic HE staining in mice, tissue sections were observed and histological scores showed that Q8-LM5-L and LGG could alleviate the histological injury caused by colitis in varying degrees. The contents of IL-8 and TNF- 伪 in mouse serum were determined by MPO Elisa with MPO kit. It was found that Lactobacillus Q8-LnM5-L and LGG could down-regulate the contents of MPOOIL-8 and TNF- 伪 and up-regulate the content of IL-10 in DSS stimulated mice. The gene expression of TLR4 and TNF- 伪 was measured by RT-PCR. The results showed that the three strains of Lactobacillus could inhibit TNF- 伪 secreted by DSS in mice at the gene level, and TLR4 receptor played a key role in the regulation of intestinal inflammation by Lactobacillus. These results suggest that Lactobacillus can significantly regulate enteritis in vivo, and the regulatory effect is different between strains and concentrations.
【学位授予单位】：哈尔滨工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62;TS201.3

【相似文献】