本文選題：乳酸桿菌 + 腸道炎癥　； 參考：《哈爾濱工業(yè)大學(xué)》2017年碩士論文

【摘要】：乳酸桿菌屬于腸道正常菌群,參與調(diào)節(jié)機(jī)體免疫應(yīng)答,以維持腸道微生態(tài)的平衡。越來(lái)越多的研究發(fā)現(xiàn),乳酸桿菌作為一種益生菌,具有多種免疫調(diào)節(jié)活性。體內(nèi)和體外實(shí)驗(yàn)都證實(shí)乳酸桿菌能夠調(diào)節(jié)腸炎。但是,目前關(guān)于乳酸桿菌對(duì)腸炎的調(diào)節(jié)作用同時(shí)進(jìn)行體內(nèi)和體外驗(yàn)證研究較少。因此本課題以脂多糖(LPS)作為炎性刺激物,研究從L.paracasei subsp.paracasei M5-L、L.casei Q8-L、L.rhamnosus J10-L和LGG對(duì)HT-29細(xì)胞炎性因子調(diào)節(jié)作用,并為動(dòng)物實(shí)驗(yàn)篩選出對(duì)腸炎抑制效果更為顯著的乳桿菌。隨后進(jìn)行動(dòng)物實(shí)驗(yàn)驗(yàn)證,動(dòng)物實(shí)驗(yàn)中以葡聚糖硫酸鈉(DSS)作為炎性刺激物,建立潰瘍性腸炎模型,研究乳桿菌對(duì)小鼠潰瘍性腸道炎癥的調(diào)節(jié)作用。體外實(shí)驗(yàn)首先對(duì)四株乳桿菌J10-L、Q8-L、M5-L和LGG進(jìn)行計(jì)數(shù),利用MTT實(shí)驗(yàn)確定后續(xù)實(shí)驗(yàn)條件為L(zhǎng)PS作用濃度為5μg/m L,乳酸桿菌與細(xì)胞的感染復(fù)數(shù)為1:1,同時(shí)證實(shí)了選定的四株乳桿菌都能夠抑制LPS對(duì)HT-29細(xì)胞增殖的影響,但是不同菌株在不同時(shí)間對(duì)細(xì)胞增殖的影響不同。通過(guò)NO試劑盒測(cè)定細(xì)胞上清液中NO,ELISA試劑盒測(cè)定細(xì)胞上清液中IL-8和TNF-α含量的檢測(cè),證明四株乳酸桿菌都能夠明顯抑制LPS刺激細(xì)胞過(guò)度分泌NO和TNF-α,并且這種抑制作用存在菌株差異性。但是對(duì)IL-8的下調(diào)作用并不顯著。體外細(xì)胞實(shí)驗(yàn)結(jié)果表明乳桿菌J10-L對(duì)腸炎的調(diào)節(jié)作用效果不顯著,并且四株菌株預(yù)防組效果相對(duì)于治療組和競(jìng)爭(zhēng)組效果更顯著。根據(jù)體外細(xì)胞實(shí)驗(yàn)結(jié)果篩選出對(duì)腸道炎癥因子抑制效果更顯著的乳酸桿菌Q8-L、M5-L和LGG進(jìn)行小鼠腸炎預(yù)防實(shí)驗(yàn)。體內(nèi)小鼠實(shí)驗(yàn)通過(guò)預(yù)實(shí)驗(yàn)確定用2%DSS來(lái)誘導(dǎo)腸炎模型,并在誘導(dǎo)小鼠腸炎模型前用乳桿菌濃度為108 cfu/m L和109 cfu/m L的乳桿菌Q8-L、M5-L和LGG灌胃4周。養(yǎng)鼠期間每天測(cè)定小鼠的體重并根據(jù)體重減輕情況和血便情況對(duì)小鼠進(jìn)行DAI評(píng)分,DAI評(píng)分表明三株乳桿菌能不同程度的減輕小鼠患病癥狀。隨后通過(guò)小鼠臟器重量和結(jié)腸長(zhǎng)度表明三株乳酸桿菌均能腸炎引起的脾重量增加和結(jié)腸變短變輕狀況。對(duì)小鼠結(jié)腸進(jìn)行HE染色后,觀察組織切片并且進(jìn)行組織學(xué)評(píng)分證實(shí)Q8-L、M5-L和LGG能不同程度的緩解腸炎帶來(lái)的組織學(xué)損傷狀況。通過(guò)MPO試劑盒測(cè)定小鼠結(jié)腸中的MPO,ELISA法測(cè)定小鼠血清中IL-8和TNF-α含量,發(fā)現(xiàn)乳酸桿菌Q8-L、M5-L和LGG都能下調(diào)DSS刺激的小鼠產(chǎn)生的MPO,IL-8和TNF-α含量,上調(diào)IL-10含量。用RT-PCR測(cè)定TLR4和TNF-α的基因表達(dá)量,在基因水平上說(shuō)明三株乳酸桿菌能抑制DSS刺激小鼠分泌的TNF-α,并且TLR4受體在乳桿菌調(diào)節(jié)腸道炎癥時(shí)起到關(guān)鍵作用。這些結(jié)果表明乳桿菌能夠顯著調(diào)節(jié)體內(nèi)腸炎,并且調(diào)節(jié)作用存在菌株和濃度差異性。
[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- 偽 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- 偽 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- 偽 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- 偽 and up-regulate the content of IL-10 in DSS stimulated mice. The gene expression of TLR4 and TNF- 偽 was measured by RT-PCR. The results showed that the three strains of Lactobacillus could inhibit TNF- 偽 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.
【學(xué)位授予單位】：哈爾濱工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574.62;TS201.3

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