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双歧杆菌对应激大鼠肠道菌群及促肾上腺皮质激素释放激素的影响

Influence of Bifidobacterium on the Intestinal Microflora and Corticotropin-releasing Factor in Rats Following Chronic Psychological Stress

作者: 专业:药物分析学 导师:付蕾 年度:2010 学位:硕士  院校: 郑州大学

Keywords

Probiotics, Chronic psychological stress, Intestinal permeability, CRF, Intestinal microflora

        研究背景和目的益生菌是人类肠道的原籍菌,在维持宿主胃肠正常生理功能中发挥重要作用。Zairian M等研究证实益生菌能够提高应激大鼠肠道屏障功能、阻断细菌移位。在种类繁多的益生菌中,双歧杆菌作为人类肠道最早出现的益生菌极具研究价值。它不仅能够调节肠道菌群失调,而且能够显著改善肠易激综合症(IBS)和炎症性肠病(IBD)患者的相关症状。益生菌所独有的生物学特点和肠道黏膜免疫反应的特异性,使肠道益生菌疗法作为治疗胃肠道疾病的一种新疗法,日益受到重视。思密达(蒙脱石散)是一种粘膜屏障剂,临床上常用来治疗腹泻。尽管大量的数据显示益生菌能够调节肠道免疫、阻断病原菌粘附提高粘膜屏障功能,但其在应激所导致的胃肠道疾病中的治疗作用研究较少。目前尚未见到长双歧杆菌对应激大鼠肠道功能的调节作用的报道。在本实验中我们拟以避水试验模拟环境和心理压力,观察在应激条件下大鼠肠道生理生化指标的变化,通过长双歧杆菌、思密达进行干预,观察二者对应激条件下大鼠肠道功能的调节作用,以及二者有无协同治疗作用,从而为临床诊断和治疗提供实验依据。方法雌性清洁级SD大鼠50只随机分为5组:正常对照组、压力试验组、双歧杆菌干预组、思密达干预组、双歧杆菌+思密达共同干预组。采用WAS (water avoidance stress)避水试验构建大鼠应激模型,应激试验为7d。1.应激试验结束后,以三糖为探针,留取大鼠24h尿液,衍生化毛细管气相色谱法(CCGC)测定大鼠尿液中两种糖的浓度,以三氯蔗糖/甘露醇(S/M)评价大鼠肠道通透性;2.取大鼠新鲜粪便,用选择性培养基平皿计数法检测大鼠粪便菌群中几种代表性菌种的数量;3.取肠系膜淋巴结(MLN)培养后测定细菌移位率;4.通过从屈氏韧带下至回盲部全部小肠的病理切片,观察肠粘膜的损伤情况;5.用酶联免疫法(ELISA)测定大鼠血清中促肾上腺皮质激素释放激素(CRF)和促肾上腺皮质激素(ACTH)的含量。结果1.与正常对照组相比,压力试验组大鼠粪便中以大肠杆菌为代表的条件致病菌数量增多(7.078±0.229 vs 7.347±0.277,P<0.05),24h尿液中甘露醇量升高(4.718%±0.399% vs 5.097%±0.453%,P<0.05),MLN细菌移位率升高(10%vs 40%,P<0.05);血清CRF (267.0±32.3 vs 300.8±34.3, pg·mL-1, P<0.05), ACTH浓度升高(5.68±0.799 vs 6.79±0.651, pg·mL-1, P<0.05)。2.与压力试验组相比,双歧杆菌干预组表现为大肠杆菌数量显著下降(7.347±0.277 vs 7.044±0.281,P<0.05),类杆菌数量显著下降(9.485±0.306 vs9.075±0.393,P<0.05),MLN细菌移位率下降(40% vs 10%,P<0.05),ACTH水平下降(6.79±0.651 vs 5.92±0.477, pg·mL-1,P<0.05);思密达组大肠杆菌数量显著下降(7.347±0.277 vs 7.054±0.302,P<0.05),MLN细菌移位率显著下降(40% vs 20%,P<0.05)。3.长双歧杆菌与思密达共同干预后,与长双歧杆菌、思密达单独干预组相比在肠道菌群及血清CRF的调节上无显著差异。结论1.在慢性应激条件下,大鼠肠道出现了以条件致病菌增多为特征的肠道菌群紊乱,MLN细菌移位率升高,肠道通透性升高,血清CRF升高,说明大鼠肠道屏障功能受损,神经内分泌处于应激状态。2.长双歧杆菌和思密达干预后,条件致病菌数量显著下降,血清CRF或ACTH均有显著下降,MLN细菌移位率显著下降,说明长双歧杆菌与思密达均能够部分缓解慢性应激所导致的肠道功能紊乱。3.长双歧杆菌和思密达二者在治疗应激所导致的肠道功能紊乱中无协同作用。4. CCGC测定应激大鼠肠道通透性,方法专属性高,灵敏度高,降低了实验成本。
    Background and ObjectiveProbiotics of human intestinal play an important role in maintaining the health of the host gastrointestinal tract. Zairian M, et al indicated that probiotics can prevent chronic stress induced intestinal abnormalities and, thereby, exert beneficial effects in the intestinal tract. There has substantial research focusing on modulation of the intestinal microbiota and host inflammatory responses by probiotics. Probiotics are defined as non-pathogenic live microorganisms that can have health benefits for the host. Such microorganisms have been used extensively for both the prevention and treatment of various inflammatory and infectious intestinal disorders, including IBD, IBS. Therefore, attention has been focused on probiotics therapeutics. Smecta (montmorillonite powder) is a mucosal barrier agent, used clinically to treat diarrhea.Despite large amount of data shows probiotics can regulate intestinal immunity, block pathogen adhesion to improve mucosal barrier function, but there were few studies related on the probiotics treating stress-related illness. Using Bifidobacterium longum to regulate intestinal function under psychological stress has not been reported yet. In this study, we intend to use water avoidance stress (WAS) to simulate environment and psychological pressure. The aim of this study is to determine the influence of chronic psychological stress on intestinal microflora and corticotropin-releasing factor (CRF) on rats and the protective effects of Bifidobacterium longum and Smecta.MethodsFifty female Sprague Dawley rats were randomly and equally divided into five groups:normal group, stress group, Bifidobacterium group, Smecta group, Bifidobacterium plus Smecta group. All these groups were subjected to either water avoidance stress (WAS) or normal condition for tow hours per day for 7 consecutive days.1. The intestinal permeability in vivo was evaluated by measuring urinary sucralose and other sugar probes including lactulose and mannitol using capillary column gas chromatography (CCGC) method;2. Some representative genera of gut flora in their feces were detected by using selected culture media plate counting method;3. The mesenteric lymph nodes (MLN) were cultured to determine the bacterial translocation;4. The small intestine between ligament of Treitz and ileocecal valve was cuted down for biopsy to observe mucosal injury;5. The contents of CRF and adrenocorticotrophic hormone (ACTH) of their blood sera were determined by enzyme-linked immunoad sorbent assay (ELISA).Results1. Compared with the normal group, the amount of Escherichia coli in stress group increased significantly (7.078±0.229 vs 7.347±0.277, P<0.05); the 24h’s mannitol concentration (4.718%<0.399% vs 5.097%±0.453%, P<0.05) and MLN bacterial translocation of stress group increased significantly (10% vs 40%, P<0.05); the CRF (267.0±32.3 vs 300.8±34.3, pg·mL-1, P<0.05) and ACTH (5.68±0.799 vs 6.79±0.651, pg·mL-1, P<0.05) levels of stress group increased significantly.2. In comparison with the stress group, the amount of Escherichia coli (7.347±0.277 vs 7.044±0.281, P<0.05), bacteroids (9.485±0.306 vs 9.075±0.393, P<0.05) and ACTH level (6.79±0.651 vs 5.92±0.477, pg·mL-1, P<0.05) in Bifidobacterium intervention group decreased significantly; the amount of Escherichia coli (7.347±0.277 vs 7.054±0.302, P<0.05) and MLN of bacterial translocation (40% vs 20%, P<0.05) in Smecta group decreased significantly.3. Compared with Bifidobacterium longum or Smecta used only group, there was little change in Bifidobacterium longum and Smecta group.Conclusions1. The amount of Escherichia coli increased and intestinal microfloradisturbance occurred; MLN bacterial translocation occurred, intestinal permeability increased and the blood sera content of CRF was increased significantly in rats after suffering from chronic psychological stress.2. After beening treated with Bifidobacterium longum and Smecta, The amount of Escher-ichia coli decreased, the CRF and ACTH levels decreased, MLN bacterial translocati-on decreased, Bifidobacterium longum and Smecta could alleviate the gut microflora disturbance caused by the chronic psychological stress and partly restore its intestinal barrier function.3. In treatment of intestinal dysfunction caused by chronic psychological stress, Bifidobacterium longum and Smecta are not in synergies.4. CCGC can be used to determine the intestinal permeability. This method was higher specificity, higher sensitivity and cloud lower the cost of experiment.
        

双歧杆菌对应激大鼠肠道菌群及促肾上腺皮质激素释放激素的影响

摘要4-6
Abstract6-8
目录9-11
中英文缩略词对照表11-12
1 正文12-38
    1.1 前言12-14
    1.2 材料14-17
        1.2.1 实验动物14
        1.2.2 主要试剂14-15
        1.2.3 主要仪器15
        1.2.4 试剂配制15-17
    1.3 方法17-25
        1.3.1 长双歧杆菌的分离与鉴定17
        1.3.2 实验分组及应激模型的建立17-18
        1.3.3 肠道通透性测定18-24
        1.3.4 血清中CRF、ACTH测定24
        1.3.5 粪便菌群分析24
        1.3.6 细菌移位24
        1.3.7 肠道组织学24-25
    1.4 结果25-29
        1.4.1 长双歧杆菌的鉴别25
        1.4.2 长双歧杆菌对应激大鼠肠道菌群的影响25-26
        1.4.3 长双歧杆菌对应激大鼠细菌移位的影响26
        1.4.4 长双歧杆菌对应激大鼠肠道通透性的影响26-28
        1.4.5 长双歧杆菌对应激大鼠血清CRF、ACTH的影响28
        1.4.6 长双歧杆菌对应激大鼠肠道组织的影响28-29
    1.5 讨论29-38
        1.5.1 动物模型的建立30-31
        1.5.2 应激相关性肠病的发生机制31-32
        1.5.3 CCGC法测定肠道通透性32-34
        1.5.4 长双歧杆菌对应激大鼠肠道菌群的调节作用34-35
        1.5.5 长双歧杆菌对应激相关性肠病的调节作用35-38
结论38-39
参考文献39-44
附图44-45
2 综述45-55
    2.1 CRF系统及其信号传导46
    2.2 胃肠道的免疫系统46-48
    2.3 应激和肠道炎症48-49
    2.4 应激与菌群失调49-50
    2.5 益生菌对应激相关性肠病的调节作用50-54
        2.5.1 益生菌简介50-51
        2.5.2 益生菌的益生保健和临床治疗作用51-53
        2.5.3 益生菌对压力相关性肠病的调节作用53-54
    2.6 小结54-55
参考文献55-60
个人简历60
在学期间发表的学术论文与研究成果60-61
致谢61
        下载全文需61


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