乳酸杆菌的分离鉴定及其对炎症反应的免疫调节作用的研究

发布时间：2018-03-02 08:32

本文关键词： 嗜酸乳酸杆菌副干酪乳酸杆菌促炎因子 NF-κB 吞噬作用　出处：《南京医科大学》2012年博士论文　论文类型：学位论文

【摘要】：背景：乳酸杆菌种类繁多，属肠道正常菌群，是益生菌的重要成员之一，能够维持机体微生态平衡，并对固有免疫和适应性免疫系统的多种组分具有调节活性。但乳酸杆菌的免疫调节作用有菌株特异性，而且机制尚不十分清楚。目的：筛选从健康人肠道分离的可作为候选益生菌的乳酸杆菌，研究其对单核细胞炎症因子的产生和T细胞分化方向的免疫调节作用，并从信号转导的分子水平阐述乳酸杆菌的抗炎机制，指导将乳酸杆菌应用于临床疾病的治疗。方法：一、用WHO推荐的方法鉴定和筛选分离得到的具有潜在益生菌特性的乳酸杆菌：以近期未服用抗生素和乳酸杆菌制剂的健康志愿者粪便为标本，在MRS琼脂平板上以厌氧培养分离乳酸杆菌；取可能的乳酸杆菌菌落，通过革兰染色、触酶试验、API50CHL生化反应系统、随机扩增多态性DNA标记（RAPD）技术进行鉴定。二、体外实验中用乳酸杆菌的活菌、灭活的菌体及细菌基因组DNA分别刺激PBMCs，观察PBMCs表达的细胞因子谱的变化，并分别用脂多糖（LPS）和抗-CD3单抗活化PBMCs中的单核细胞和T细胞，同样在细胞培养体系中加乳酸杆菌或其DNA，观察其对LPS激发的单核细胞促炎因子的释放以及抗-CD3抗体活化的T细胞中Th1与Th2型细胞因子释放的影响，初步分析所分离的两株乳酸杆菌对炎症反应和T细胞分化方向的免疫调节作用；培养上清中细胞因子检测采用ELISA法。三、体外实验中用LPS活化PMA诱导分化的THP-1细胞系，观察乳酸杆菌对LPS诱导的炎症因子及炎症信号分子的影响和THP-1细胞对乳酸杆菌的吞噬消化作用，阐明乳酸杆菌抗炎作用的可能分子机制；实验中，炎症因子的检测采用ELISA法，TNF-αmRNA检测采用RT-PCR法，炎症信号通路信号分子IκB检测采用Westernblotting，对NF-κBp65采用免疫荧光染色，激光共聚焦显微镜观察和核内定量检测，THP-1细胞对乳酸杆菌的吞噬作用吉姆萨染色光镜观察，，荧光染色激光共聚焦显微镜观察及流式细胞术进行分析。结果：一、MRS琼脂平板上获得两株候选菌株，菌落光滑、圆型、边缘整齐、表面突起、湿润细腻；革兰染色阳性，细长杆状；触酶试验阴性；经API50CHL生化反应系统鉴定，一株为嗜酸乳酸杆菌，鉴定百分率为94.5%，命名为C44，另一株为副干酪乳酸杆菌，鉴定百分率为95.5%，命名为C48。RAPD鉴定与相应的标准株基本一致，确定分离的乳酸杆菌分别是嗜酸乳酸杆菌（L.acidophilus）和副干酪乳酸杆菌（L.paracacei）。该两株乳酸杆菌对胆汁和胃酸有良好的耐受性，对病原菌有一定的抑制作用。二、两株乳酸杆菌直接刺激PBMCs诱导低水平促炎因子TNF-α、IL-6、IL-8、IL-12p70；诱导较高水平Th1型细胞因子IFN-γ产生；三、在LPS活化的PBMCs中，两株乳酸杆菌能显著降低LPS诱导的炎症因子TNF-α、IL-6的释放，而且以C48（副干酪乳酸杆菌）的抗炎作用更为突出；在抗-CD3单抗活化的PBMCs中，则能下调Th2型的细胞因子IL-4、IL-5、IL-10、IL-13的水平；上调Th1型细胞因子IFN-γ的表达，这种诱导Th1分化的作用以C44（嗜酸乳酸杆菌）更为突出。四、乳酸杆菌的活菌与热灭活菌作用基本一致，基因组DNA有更强的抗炎作用。五、两株乳酸杆菌可以抑制LPS活化的PMA诱导分化的THP-1细胞产生促炎因子TNF-α和IL-12，同时能下调IκB的磷酸化水平和NF-κBp50/p65的核转位。六、嗜酸乳酸杆菌比副干酪乳酸杆菌更容易被PMA诱导分化的THP-1细胞吞噬和消化。结论：一、分离到两株乳酸杆菌分别鉴定为嗜酸乳酸杆菌（C44）和副干酪乳酸杆菌（C48），并能顺利进入肠道，具备作为益生菌的条件。二、两株乳酸杆菌均具有免疫调节作用，表现为能降低单核巨噬细胞的炎症反应，促进巨噬细胞的吞噬功能，同时能诱导Th1分化，抑制Th2优势，因此具有抗炎和抗过敏的临床应用潜能。三、乳酸杆菌的抗炎作用是通过抑制IκB的磷酸化，抑制NF-κB的活化转位，进而减少TNF-αmRNA的转录，降低了促炎性细胞因子的合成来实现的。四、乳酸杆菌的免疫调节作用具有种的差异性，同时对吞噬细胞的吞噬消化作用的抵抗力也有种的差异；由于吞噬消化作用的差异，两株乳酸杆菌与巨噬细胞接触后可能通过菌体上的不同成分与巨噬细胞上的模式识别受体结合，激发不同的信号传导途径。
[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.

【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R151

【共引文献】