鼠李糖乳杆菌LV108对免疫低下大鼠肠道菌群及代谢的影响研究

发布时间：2018-04-29 14:08

本文选题：乳酸菌 + 免疫调节　；参考：《扬州大学》2016年硕士论文

【摘要】：乳酸菌是人体肠道中重要的有益菌,可以与肠道粘膜免疫系统相互作用,在抑制肠道致病菌、调节机体免疫功能、维持肠道微生态平衡等方面发挥了重要作用。当肠道菌群的平衡受到破坏时,宿主的正常代谢就会受到扰乱,甚至导致疾病。本文对源于广西巴马和新疆和田长寿人群粪便中的乳酸菌进行体外分离筛选,研究其在体内的免疫调节作用,并利用实时荧光定量PCR技术和核磁共振技术探讨其对免疫低下大鼠肠道菌群及宿主代谢的影响,从而为益生菌干预亚健康膳食提供助益。(1)从广西巴马和新疆和田长寿人群粪便中分离乳酸菌,并研究其体外疏水能力、对细胞黏附能力、耐酸耐胆盐能力及抑菌特性和耐药性。结果表明,分离获得的153株乳酸菌中,10株乳酸菌的表面疏水能力较好、对细胞黏附能力较强；进一步研究发现,10株乳酸菌中菌株PW1、LV108、XJH301在pH2.0的人工胃液中培养2h后的存活率分别为34.08%、61.35%、47.15%,在人工肠液中培养4h后的存活率分别为61.42%、86.03%、55.03%,在浓度0.3%的胆盐MRS培养基中培养24h后的存活率分别为49.36%、45.24%、37.19%,显著高于其它菌株(P0.05)；对大肠杆菌的抑菌圈分别为16.76mm、20.49mm、14.35mm,对沙门氏菌的抑菌圈分别为13.23mm、17.19mm、10.63mm,对金黄色葡萄球菌的抑菌圈分别为1176mm、14.01mm、10.10mm;菌株LV108对利福平、四环素、头孢唑啉、氯霉素、头孢拉定五种抗生素均敏感,对链霉素中度敏感,耐药性低于其他菌株；经API及16SrDNA测序将LV108菌株鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。(2)以左旋咪唑作为阳性对照,通过连续3天腹腔注射40mg/kg环磷酰胺的方法建立免疫低下大鼠模型。在建模期间,大鼠体重增加缓慢,胸腺生长受到抑制。血清中的IL-2、IgG、IgA含量、溶血素含量显著降低(P0.05)；大鼠肝、脾巨噬细胞的廓清指数K、吞噬指数α显著低于对照组(P0.05),脾淋巴细胞数量显著下降(P0.05),建模成功。干预4周后发现,L. rhamnosus LV108菌体悬浮液及其发酵乳能显著提高大鼠脾脏指数和胸腺指数(P0.05)；同时还能显著增加大鼠血清中IL-2、ⅠgG含量(P0.05)；刺激B淋巴细胞分化增殖,提高血清中的溶血素含量；显著提高免疫低下大鼠的肝、脾巨噬细胞廓清指数K、吞噬指数α(P0.05),提高了大鼠单核巨噬细胞系统功能；促进大鼠脾淋巴细胞增殖,进而参与机体脾淋巴细胞的免疫应答。(3)利用实时荧光定量PCR法研究大鼠肠道菌群的变化。结果表明,与对照组相比,免疫低下大鼠肠道菌群发生了变化,其中乳酸菌和双歧杆菌的含量显著降低(P0.05)而肠杆菌和肠球菌的含量显著增加(P0.05)。干预4周后发现,L. rhamnosus LV108菌体悬浮液及其发酵乳能显著降低大鼠肠道中肠杆菌、肠球菌的含量(P0.05),而显著增加大鼠肠道中乳酸菌及双歧杆菌含量(P0.05)。L. rhamnosus LV108发酵乳对大鼠肠道菌群的改善幅度要大于L. rhamnosus LV108菌悬液,表明活的LV108菌体既能通过自身发挥作用,还能通过其代谢产物来改善肠道菌群。相关性分析发现,大鼠肠道内乳酸菌的含量同其血清中TNF-a及ⅠgA的水平呈显著正相关(P0.05)。(4)利用核磁共振技术研究大鼠肠道代谢产物的变化。结果发现,与对照组相比,免疫低下大鼠粪便代谢产物中异亮氨酸、丙氨酸、丁酸、琥珀酸、磷酸肌酸、氧化三甲胺、牛磺酸、β-葡萄糖、α-葡萄糖、尿嘧啶等成分含量降低。L. rhamnosus LV108菌体悬浮液及发酵乳对大鼠进行4周干预后,增加了大鼠粪便代谢产物中异亮氨酸、丙氨酸、赖氨酸等氨基酸的含量,改善了大鼠的氨基酸代谢和能量代谢；增加了乳酸、琥珀酸等有机酸的含量,抑制了肠道中致病菌的生长；增加了α-葡萄糖、N-乙酰糖蛋白、TMA、β-葡萄糖等成分的含量,改善了机体肠道菌群的代谢。
[Abstract]:Lactic acid bacteria, an important beneficial bacteria in the intestinal tract, can interact with the intestinal mucosal immune system. It plays an important role in inhibiting the intestinal pathogenic bacteria, regulating the immune function of the body and maintaining the intestinal microecological balance. When the balance of the intestinal flora is destroyed, the normal metabolism of the host will be disturbed and even causes the disease. In vitro isolation and screening of lactic acid bacteria derived from the feces of Guangxi Bama and Xinjiang Hetian long - lived population, the immunomodulatory effect of the lactic acid bacteria in the body was studied in this paper. The effects on intestinal microflora and host metabolites of immunocompromised rats were investigated by real time fluorescence quantitative PCR technique and nuclear magnetic resonance technique, so as to interfere with subhealth for probiotics. (1) isolation of lactic acid bacteria from the feces of Guangxi Bama and Xinjiang Hetian long - lived population, and study the hydrophobic ability in vitro, cell adhesion, acid resistance, salt tolerance and antimicrobial resistance and resistance. The results showed that of the 153 Lactobacillus Isolated, 10 Lactobacillus strains had better surface hydrophobicity and cell adhesion energy. The survival rate of the 10 strains of lactic acid bacteria PW1, LV108 and XJH301 in the artificial gastric juice of pH2.0 was 34.08%, 61.35%, 47.15% respectively. The survival rate of 4H in the artificial intestinal juice was 61.42%, 86.03%, 55.03%, respectively, and the survival rate of 24h in the bile salt MRS culture medium with the concentration of 0.3% was 49.36%, respectively. 45.24%, 37.19%, significantly higher than the other strains (P0.05), the Bacteriostasis of Escherichia coli were 16.76mm, 20.49mm, 14.35mm, and the bacteriostatic ring of Salmonella was 13.23mm, 17.19mm, 10.63mm, respectively, 1176mm, 14.01mm, 10.10mm for Staphylococcus aureus, and LV108 against Li Fuping, tetracycline, cefazolin, chloramphenicol, head. Five kinds of antibiotics were sensitive, and streptomycin was moderately sensitive and drug resistance was lower than that of other strains. LV108 was identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus) by API and 16SrDNA sequencing. (2) levamisole was used as a positive control, and the immunocompromised rat model was established by intraperitoneal injection of 40mg/kg cyclophosphamide for 3 days. During the modeling period, the body weight increased slowly and the thymus growth was inhibited. The content of IL-2, IgG, IgA in the serum, the content of the hemolysin decreased significantly (P0.05), the clearance index of the liver and spleen macrophages in rats was K, the phagocytic index alpha was significantly lower than that of the control group (P0.05), the number of spleen lymphocytic cells decreased significantly (P0.05), and the modeling was successful. After 4 weeks of intervention, L. was found, L. Rhamnosus LV108 mycelium suspension and its fermented milk could significantly increase the spleen index and thymus index (P0.05) in rats, and also significantly increase the serum IL-2, gG content (P0.05), stimulate the proliferation of B lymphocytes and increase the content of hemolysin in the serum, and significantly improve the liver and spleen macrophage clearance finger in the immunocompromised rats. K, phagocytic index alpha (P0.05), increased the function of mononuclear macrophage system in rats, promoted the proliferation of spleen lymphocyte in rats, and then participated in the immune response of the spleen lymphocyte of the body. (3) the changes of intestinal flora in rats were studied by real-time fluorescence quantitative PCR. The results showed that the intestinal flora of immunocompromised rats was changed compared with the control group. The content of lactic acid bacteria and bifidobacteria significantly decreased (P0.05) and the content of enterobacteria and Enterococcus increased significantly (P0.05). After 4 weeks of intervention, it was found that L. rhamnosus LV108 mycelium suspension and its fermented milk could significantly reduce intestinal bacilli and Enterococcus (P0.05) in rats, and significantly increase the lactic acid bacteria and bifidobacteria in the intestine of rats. The improvement of intestinal microflora by bacilli content (P0.05).L. rhamnosus LV108 is greater than that of L. rhamnosus LV108 bacteria suspension. It shows that the living LV108 bacteria can not only play its role, but also improve the intestinal microflora through its metabolites. The correlation analysis shows that the content of lactic acid bacteria in the intestine of rats is the same as that of the serum in the rat. There was a significant positive correlation with the level of I gA (P0.05). (4) the changes in intestinal metabolites of rats were studied by NMR. Results showed that compared with the control group, isoleucine, alanine, butyric acid, succinic acid, creatine phosphocreatine, trimethylamine, taurine, beta glucose, alpha glucose, uracil, and so on were compared with the control group. The content of.L. rhamnosus LV108 bacterial suspension and fermented milk was reduced for 4 weeks. The contents of isoleucine, alanine, lysine and other amino acids were increased in rats' fecal metabolites, which improved the amino acid metabolism and energy metabolism in rats, increased the content of lactic acid, succinic acid and other organic acids, and inhibited the intestinal tract. The growth of pathogenic bacteria increased the content of alpha glucose, N- acetylglycoprotein, TMA, beta glucose and other components, and improved the metabolism of intestinal flora.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R151

【参考文献】