本文關(guān)鍵詞： 嗜酸乳酸桿菌 副干酪乳酸桿菌 促炎因子 NF-κB 吞噬作用　出處：《南京醫(yī)科大學(xué)》2012年博士論文　論文類型：學(xué)位論文

【摘要】：背景：乳酸桿菌種類繁多，屬腸道正常菌群，是益生菌的重要成員之一，能夠維持機(jī)體微生態(tài)平衡，并對(duì)固有免疫和適應(yīng)性免疫系統(tǒng)的多種組分具有調(diào)節(jié)活性。但乳酸桿菌的免疫調(diào)節(jié)作用有菌株特異性，而且機(jī)制尚不十分清楚。 目的：篩選從健康人腸道分離的可作為候選益生菌的乳酸桿菌，研究其對(duì)單核細(xì)胞炎癥因子的產(chǎn)生和T細(xì)胞分化方向的免疫調(diào)節(jié)作用，并從信號(hào)轉(zhuǎn)導(dǎo)的分子水平闡述乳酸桿菌的抗炎機(jī)制，指導(dǎo)將乳酸桿菌應(yīng)用于臨床疾病的治療。 方法：一、用WHO推薦的方法鑒定和篩選分離得到的具有潛在益生菌特性的乳酸桿菌：以近期未服用抗生素和乳酸桿菌制劑的健康志愿者糞便為標(biāo)本，在MRS瓊脂平板上以厭氧培養(yǎng)分離乳酸桿菌；取可能的乳酸桿菌菌落，通過(guò)革蘭染色、觸酶試驗(yàn)、API50CHL生化反應(yīng)系統(tǒng)、隨機(jī)擴(kuò)增多態(tài)性DNA標(biāo)記（RAPD）技術(shù)進(jìn)行鑒定。二、體外實(shí)驗(yàn)中用乳酸桿菌的活菌、滅活的菌體及細(xì)菌基因組DNA分別刺激PBMCs，觀察PBMCs表達(dá)的細(xì)胞因子譜的變化，并分別用脂多糖（LPS）和抗-CD3單抗活化PBMCs中的單核細(xì)胞和T細(xì)胞，同樣在細(xì)胞培養(yǎng)體系中加乳酸桿菌或其DNA，觀察其對(duì)LPS激發(fā)的單核細(xì)胞促炎因子的釋放以及抗-CD3抗體活化的T細(xì)胞中Th1與Th2型細(xì)胞因子釋放的影響，初步分析所分離的兩株乳酸桿菌對(duì)炎癥反應(yīng)和T細(xì)胞分化方向的免疫調(diào)節(jié)作用；培養(yǎng)上清中細(xì)胞因子檢測(cè)采用ELISA法。三、體外實(shí)驗(yàn)中用LPS活化PMA誘導(dǎo)分化的THP-1細(xì)胞系，觀察乳酸桿菌對(duì)LPS誘導(dǎo)的炎癥因子及炎癥信號(hào)分子的影響和THP-1細(xì)胞對(duì)乳酸桿菌的吞噬消化作用，闡明乳酸桿菌抗炎作用的可能分子機(jī)制；實(shí)驗(yàn)中，炎癥因子的檢測(cè)采用ELISA法，TNF-αmRNA檢測(cè)采用RT-PCR法，炎癥信號(hào)通路信號(hào)分子IκB檢測(cè)采用Westernblotting，對(duì)NF-κBp65采用免疫熒光染色，激光共聚焦顯微鏡觀察和核內(nèi)定量檢測(cè)，THP-1細(xì)胞對(duì)乳酸桿菌的吞噬作用吉姆薩染色光鏡觀察，，熒光染色激光共聚焦顯微鏡觀察及流式細(xì)胞術(shù)進(jìn)行分析。 結(jié)果：一、MRS瓊脂平板上獲得兩株候選菌株，菌落光滑、圓型、邊緣整齊、表面突起、濕潤(rùn)細(xì)膩；革蘭染色陽(yáng)性，細(xì)長(zhǎng)桿狀；觸酶試驗(yàn)陰性；經(jīng)API50CHL生化反應(yīng)系統(tǒng)鑒定，一株為嗜酸乳酸桿菌，鑒定百分率為94.5%，命名為C44，另一株為副干酪乳酸桿菌，鑒定百分率為95.5%，命名為C48。RAPD鑒定與相應(yīng)的標(biāo)準(zhǔn)株基本一致，確定分離的乳酸桿菌分別是嗜酸乳酸桿菌（L.acidophilus）和副干酪乳酸桿菌（L.paracacei）。該兩株乳酸桿菌對(duì)膽汁和胃酸有良好的耐受性，對(duì)病原菌有一定的抑制作用。二、兩株乳酸桿菌直接刺激PBMCs誘導(dǎo)低水平促炎因子TNF-α、IL-6、IL-8、IL-12p70；誘導(dǎo)較高水平Th1型細(xì)胞因子IFN-γ產(chǎn)生；三、在LPS活化的PBMCs中，兩株乳酸桿菌能顯著降低LPS誘導(dǎo)的炎癥因子TNF-α、IL-6的釋放，而且以C48（副干酪乳酸桿菌）的抗炎作用更為突出；在抗-CD3單抗活化的PBMCs中，則能下調(diào)Th2型的細(xì)胞因子IL-4、IL-5、IL-10、IL-13的水平；上調(diào)Th1型細(xì)胞因子IFN-γ的表達(dá)，這種誘導(dǎo)Th1分化的作用以C44（嗜酸乳酸桿菌）更為突出。四、乳酸桿菌的活菌與熱滅活菌作用基本一致，基因組DNA有更強(qiáng)的抗炎作用。五、兩株乳酸桿菌可以抑制LPS活化的PMA誘導(dǎo)分化的THP-1細(xì)胞產(chǎn)生促炎因子TNF-α和IL-12，同時(shí)能下調(diào)IκB的磷酸化水平和NF-κBp50/p65的核轉(zhuǎn)位。六、嗜酸乳酸桿菌比副干酪乳酸桿菌更容易被PMA誘導(dǎo)分化的THP-1細(xì)胞吞噬和消化。 結(jié)論：一、分離到兩株乳酸桿菌分別鑒定為嗜酸乳酸桿菌（C44）和副干酪乳酸桿菌（C48），并能順利進(jìn)入腸道，具備作為益生菌的條件。二、兩株乳酸桿菌均具有免疫調(diào)節(jié)作用，表現(xiàn)為能降低單核巨噬細(xì)胞的炎癥反應(yīng)，促進(jìn)巨噬細(xì)胞的吞噬功能，同時(shí)能誘導(dǎo)Th1分化，抑制Th2優(yōu)勢(shì)，因此具有抗炎和抗過(guò)敏的臨床應(yīng)用潛能。三、乳酸桿菌的抗炎作用是通過(guò)抑制IκB的磷酸化，抑制NF-κB的活化轉(zhuǎn)位，進(jìn)而減少TNF-αmRNA的轉(zhuǎn)錄，降低了促炎性細(xì)胞因子的合成來(lái)實(shí)現(xiàn)的。四、乳酸桿菌的免疫調(diào)節(jié)作用具有種的差異性，同時(shí)對(duì)吞噬細(xì)胞的吞噬消化作用的抵抗力也有種的差異；由于吞噬消化作用的差異，兩株乳酸桿菌與巨噬細(xì)胞接觸后可能通過(guò)菌體上的不同成分與巨噬細(xì)胞上的模式識(shí)別受體結(jié)合，激發(fā)不同的信號(hào)傳導(dǎo)途徑。
[Abstract]:Background: Lactobacillus species, belongs to the normal intestinal flora, is one of the important members of probiotics, can maintain the body micro ecological balance, and various groups of innate and adaptive immune system can regulate the activity. But the immune regulating effect of Lactobacillus strain specificity, and the mechanism is not very clear.
Objective: screening isolated from healthy human intestinal Lactobacillus as candidate probiotics, immune regulation effect on monocyte cytokine production and T cell differentiation, and expounds the anti-inflammatory mechanism of lactic acid bacteria from the molecular level of signal transduction, Lactobacillus treatment guidelines will be applied to clinical disease.
Methods: a method for identification and isolation, recommended by WHO, the potential probiotic properties of Lactobacillus in recent not taking antibiotics and Lactobacillus in healthy volunteers for fecal specimens in MRS agar with anaerobic culture of Lactobacillus Isolated from Lactobacillus colony; from May, by Gram staining, touch enzyme test, API50CHL biochemical reaction system, random amplified polymorphic DNA (RAPD) were identified. Two bacteria with lactic acid bacteria in vitro, killing the bacteria and the bacterial genomic DNA live were stimulated PBMCs, cytokine changes observed the expression of PBMCs spectrum, and were treated with lipopolysaccharide (LPS) and anti -CD3 monoclonal antibody activated monocytes and T cells in PBMCs, in the same cell culture system with lactobacillus or DNA, observe the proinflammatory cytokines on monocyte stimulated the release of LPS and anti -CD3 antibody Effect of release of Th1 and Th2 type cytokines in activated T cells and preliminary analysis of immunomodulatory effect of two Lactobacillus strains isolated on inflammatory response and T cell differentiation; detection of cytokines in the supernatant of cultured by the method of ELISA. Three, THP-1 cell line induced by the activation of PMA in differentiation of LPS in vitro, observation phagocytosis and digestion effects of lactic acid bacteria effect on LPS induced inflammatory cytokines and inflammatory signaling molecules and THP-1 cells of Lactobacillus, may elucidate the molecular mechanism of lactic acid bacteria and anti-inflammatory effects; experiment, detection of inflammatory factors by ELISA method, mRNA RT-PCR method was used to detect TNF- alpha, inflammatory signaling pathways were detected by I kappa B Westernblotting, of NF- kappa Bp65 by immunofluorescence staining, confocal microscopy and quantitative detection of THP-1 cells in the nucleus, the lactic acid bacteria phagocytosis Giemsa staining by light microscope Inspection, fluorescence staining laser confocal microscopy and flow cytometry were used for analysis.
Results: 1, MRS agar obtained two strains of candidate strains, the colony is smooth, round, neat edge, surface, moist and delicate; gram positive, slender; catalase test negative; identified by API50CHL biochemical reaction system, a strain of Lactobacillus acidophilus, the identification percentage is 94.5%, named C44, a strain of Lactobacillus paracasei, identification was 95.5%, named C48.RAPD identification and corresponding standard strains were consistent, lactobacillus were identified Lactobacillus acidophilus (L.acidophilus) and Lactobacillus paracasei (L.paracacei). The two strains of lactic acid bacteria has good tolerance to bile and gastric acid has inhibitory effects on the pathogenic bacteria. Two, two strains of Lactobacillus directly stimulate PBMCs induced by low levels of proinflammatory factor TNF- alpha, IL-6, IL-8, IL-12p70; induced higher levels of Th1 type cytokine IFN- gamma production; three, in LPS The activation of PBMCs, two strains of Lactobacillus could significantly reduce LPS induced inflammatory factor TNF- alpha, the release of IL-6, and the C48 (Lactobacillus paracasei) the anti-inflammatory effect is more prominent; in the activation of anti -CD3 monoclonal antibody PBMCs, can downregulate Th2 type cytokines IL-4, IL-5, IL-10, IL-13 the level of expression of Th1 type cytokines; IFN- gamma, Th1 induced differentiation of this role to C44 (Lactobacillus acidophilus) is more prominent. Four, live bacteria and Lactobacillus heat quenching bacteria are basically the same, genomic DNA has anti-inflammatory effects stronger. Five, two strains of milk can inhibit the activation of LPS oxydans PMA induced differentiation of THP-1 cells to produce proinflammatory cytokines of TNF- alpha and IL-12, and down-regulation of I kappa B phosphorylation and NF- kappa Bp50/p65 nuclear translocation. Six, Lactobacillus acidophilus than Lactobacillus paracasei were more likely to be PMA induced differentiation of THP-1 cells engulf and digest.
Conclusions: first, isolated two strains of lactobacillus were identified as Lactobacillus acidophilus (C44) and Lactobacillus paracasei (C48), and can successfully enter the intestinal tract, have used as probiotics. The condition of two, two strains of lactobacilli have immunomodulatory effects, as can reduce the inflammatory reaction of mononuclear macrophage promote the phagocytosis of macrophages, and can induce Th1 differentiation, inhibition of Th2 clinical application advantages, so it has potential anti-inflammatory and anti allergy. Three, anti-inflammatory effects of lactic acid bacteria by inhibiting I kappa B phosphorylation, translocation activation inhibition of NF- K B, thereby reducing the transcription of TNF- alpha mRNA, reduced the synthesis of Pro inflammatory cytokines to achieve. Four, immunomodulatory effects of lactic acid bacteria have different kinds, at the same time to ingest phagocyte resistance also have a difference; because of phagocytosis and digestion with two different strains of lactic acid After contact with macrophages, bacilli may bind to the pattern recognition receptors on the macrophages by different components on the bacteria and stimulate different signal transduction pathways.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R151

【共引文獻(xiàn)】

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