氨溴索抗流感嗜血杆菌生物膜机制的初步研究

发布时间：2018-07-12 10:27

本文选题：流感嗜血杆菌 + 生物膜　；参考：《河北北方学院》2015年硕士论文

【摘要】：细菌生物膜(biofilm,BF)是细菌形成的一种与游离细菌相对应的生长方式,它是一种动态的、混杂、持续变化的群落,其内部可能仅包含某一种细菌或真菌,更多时候则可能包含多种不同的微生物。它可被形容为细菌被包埋在一层由糖分和蛋白质构成的厚厚的,粘性的屏障中。这种BF屏障能保护微生物免受外界的威胁,被认为是持续性感染性疾病中产生耐药的重要因素。BF理论于1978年首次提出,现已证实了很多疾病与细菌BF的形成相关,其中包括龋齿、牙周炎、心内膜炎和肺囊性纤维化等疾病。近年来,有关BF与临床疾病的关系得到的广泛研究,现已证实,很多临床疾病与流感嗜血杆菌(Haemophilus influenzae)BF的形成有关,如:慢性阻塞性肺疾病(COPD)急性发作、肺囊性纤维化(CF)、慢性鼻-鼻窦炎(CRS)、分泌性中耳炎(OMA)、腺样体肥大等。氨溴索(Ambroxol,AMB)是一种化痰药,研究表明其对金黄色葡萄球菌、铜绿假单胞菌等的BF具有清除和破坏作用。它可使编码金黄色葡萄球菌胞外脂多糖主要成分藻酸盐合成的相关基因algD、algR、algU表达下调,而使抑制其合成的基因mucA表达上调,进而抑制其BF的形成;亦能够通过抑制编码铜绿假单胞菌密度感应(quorum sensing,QS)系统信号分子AHL合成酶的基因las I和rhl I的表达,使信号分子AHL的合成减少,进而抑制其BF的形成。本实验室前期研究亦发现AMB对流感嗜血杆菌体外形成的BF有破坏和清除作用,且联合抗生素的使用可大大增加抗杀菌效果。但其清除和破坏作用机制尚未阐明,亦未见相关报道。本课题从流感嗜血杆菌游离态、形成BF的早期粘附阶段以及成熟的BF三个阶段入手,探究AMB抗流感嗜血杆菌BF的机制。实验选取流感嗜血杆菌标准菌株ATCC24479和临床分离的成膜能力较强的Hi23为实验菌株。首先,连续动态观察不同浓度的AMB对未形成BF的浮游态的流感嗜血杆菌的作用,对比培养基下流感嗜血杆菌生长情况,绘制其生长曲线;其次,采用半定量实验检测不同浓度的AMB对流感嗜血杆菌BF形成早期的粘附情况的影响,标本经染色、脱色等处理后,以多功能酶标仪检测其A570的OD值,并行统计分析;再次,体外建立流感嗜血杆菌成熟BF,以扫描电镜鉴定其生成情况,试剂盒法提取流感嗜血杆菌成熟BF的总RNA,微量分光光度计鉴定其纯度和浓度质量后行逆转录,再行实时荧光定量反应,比较分析不同浓度AMB作用下,流感嗜血杆菌BF形成相关基因luxS、pilA、licD的mRNA表达情况。根据不同时间流感嗜血杆菌生长情况绘制生长曲线发现,不同浓度的AMB对ATCC24479和Hi23两株菌浮游态生长影响较小,曲线未表现出明显差异。而将培养了不同时间的ATCC24479和Hi23两株菌的96孔板经结晶紫染色后以酶标仪检测,统计分析A值显示:2株6组流感嗜血杆菌经不同时间培养后,都有不同程度早期粘附的BF形成,与培养基对照组相比,不同浓度的AMB均可减少流感嗜血杆菌早期的粘附(P0.05),从而抑制其BF的进一步生成;高浓度的AMB作用更加明显,表现出浓度依赖性(P0.05)。成功于体外建立流感嗜血杆菌BF模型,并完成BF总RNA的提取、逆转录和荧光定量PCR反应等试验,实验结果分析显示:空白对照组luxS、pilA、licD基因表达最高;两实验组与对照组相比,luxS、pilA、licD基因表达量均明显减少,差异具有统计学意义(P0.05);AMB(3.75mg/ml)组与AMB(1.875mg/ml)组相比lux S、pilA、licD基因表达均减少更明显,差异具有统计学意义(P0.05)。综上实验得出:AMB对浮游态的流感嗜血杆菌的生长无明显影响;AMB可减弱流感嗜血杆菌BF形成早期的粘附作用,进而影响其BF的进一步生成;AMB可下调流感嗜血杆菌BF形成相关基因luxS、pilA、licD的表达,进而影响其形成和进一步成熟。本课题为AMB在临床的使用提供了理论基础,可以为临床治疗流感嗜血杆菌BF感染性疾病提供有益的用药指导。
[Abstract]:The bacterial biofilm (biofilm (BF) is a form of bacterial growth that corresponds to free bacteria. It is a dynamic, mixed, continuously changing community, which may contain only one bacterium or fungus, and more often contains a variety of different microorganism. It can be described as a bacterium buried in a layer of sugar and in a layer of sugar. A thick, sticky barrier made up of proteins. This BF barrier can protect microorganisms from external threats and is considered to be an important factor in resistance to persistent infectious diseases..BF theory was first proposed in 1978. Many diseases have been proved to be related to the formation of bacterial BF, including dental caries, periodontitis, endocarditis and endocarditis. The relationship between BF and clinical diseases has been widely studied in recent years. It has been confirmed that many clinical diseases are associated with the formation of Haemophilus influenzae BF, such as acute obstructive pulmonary disease (COPD), pulmonary cystic fibrosis (CF), chronic rhinosinusitis (CRS), and secretory otitis media. (OMA), adenoid hypertrophy, ambroxol (Ambroxol, AMB) is a kind of expectorant. The study shows that it has scavenging and destroying effects on BF, Staphylococcus aureus, Pseudomonas aeruginosa, and so on. It can reduce the expression of algD, algR, algU expression of the main component of the extracellular lipopolysaccharide, the main component of the extracellular lipopolysaccharide, in the alginate synthesis of Staphylococcus aureus. The expression of the gene mucA is up and then inhibits the formation of its BF, and can also reduce the synthesis of Las I and RHL I of the AHL synthase gene of the signal molecules of the quorum sensing (QS) system of Pseudomonas aeruginosa, which can inhibit the formation of the BF. BF, formed by Haemophilus influenzae in vitro, has the destruction and scavenging effect, and the use of combined antibiotics can greatly increase the antiseptic effect. However, the mechanism of its clearance and destruction has not been clarified, and no related reports have been reported. This subject starts with the free state of Haemophilus influenzae, the formation of the early adhesion stage of BF and the three stages of mature BF, and explores AMB The mechanism of anti Haemophilus influenzae BF. The experiment selected the standard strain ATCC24479 of Haemophilus influenzae and the clinically isolated Hi23 as the experimental strain. First, the effect of different concentrations of AMB on Haemophilus influenzae without BF formation was continuously dynamically observed, and the growth of Haemophilus influenzae under the culture medium was compared. Secondly, the effect of different concentrations of AMB on the early adhesion of Haemophilus influenzae BF was detected by semi quantitative test. After dyed and decolorizing, the A570 was detected by the multi-function enzyme labeling instrument, and the statistical analysis was carried out. Again, the mature BF of Haemophilus influenzae was established in vitro, and its birth was identified by scanning electron microscope. In this case, the total RNA of Haemophilus influenzae mature BF was extracted by the reagent box method. After the trace spectrophotometer was used to identify its purity and concentration quality, a real-time fluorescent quantitative reaction was performed to compare the mRNA expression of luxS, pilA, licD, related genes of Haemophilus influenzae BF, under the action of different concentrations of AMB. The growth curve of blood bacilli showed that the AMB of different concentrations had little influence on the growth of ATCC24479 and Hi23 two strains, and the curve did not show obvious difference. The 96 hole plates of ATCC24479 and Hi23 two strains of different time were detected by the enzyme labeling after crystal violet staining, and the statistical analysis A value showed that 6 groups of 6 groups of influenza were addicted to influenza. After different time culture, there were different degrees of early adherent BF formation. Compared with the culture base control group, different concentrations of AMB could reduce the early adhesion of Haemophilus influenzae (P0.05) and inhibit the further formation of its BF; the high concentration of AMB was more evident and showed a concentration dependence (P0.05). The effect was successfully established in vitro. Haemophilus influenzae BF model, and complete BF total RNA extraction, reverse transcription and fluorescence quantitative PCR reaction tests, the results of the experimental analysis showed that the blank control group luxS, pilA, licD gene expression was the highest; two the experimental group compared with the control group, luxS, pilA, licD gene expression was significantly reduced, the difference was statistically significant (P0.05); AMB (AMB) group Compared with the AMB (1.875mg/ml) group, the expression of lux S, pilA, licD gene decreased more obviously, and the difference was statistically significant (P0.05). It was concluded that AMB had no obvious effect on the growth of Haemophilus influenzae in floating state; AMB could weaken the early adhesion of Haemophilus influenzae BF, and then affect the further formation of BF. The expression of the related gene luxS, pilA, and licD of Haemophilus influenzae BF affects its formation and further maturation. This topic provides a theoretical basis for the clinical use of AMB, which can provide useful guidance for the clinical treatment of Haemophilus influenzae BF infected diseases.
【学位授予单位】：河北北方学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.5

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