本文選題：流感嗜血桿菌 + 生物膜　； 參考：《河北北方學院》2015年碩士論文

【摘要】：細菌生物膜(biofilm,BF)是細菌形成的一種與游離細菌相對應的生長方式,它是一種動態(tài)的、混雜、持續(xù)變化的群落,其內部可能僅包含某一種細菌或真菌,更多時候則可能包含多種不同的微生物。它可被形容為細菌被包埋在一層由糖分和蛋白質構成的厚厚的,粘性的屏障中。這種BF屏障能保護微生物免受外界的威脅,被認為是持續(xù)性感染性疾病中產生耐藥的重要因素。BF理論于1978年首次提出,現(xiàn)已證實了很多疾病與細菌BF的形成相關,其中包括齲齒、牙周炎、心內膜炎和肺囊性纖維化等疾病。近年來,有關BF與臨床疾病的關系得到的廣泛研究,現(xiàn)已證實,很多臨床疾病與流感嗜血桿菌(Haemophilus influenzae)BF的形成有關,如:慢性阻塞性肺疾病(COPD)急性發(fā)作、肺囊性纖維化(CF)、慢性鼻-鼻竇炎(CRS)、分泌性中耳炎(OMA)、腺樣體肥大等。氨溴索(Ambroxol,AMB)是一種化痰藥,研究表明其對金黃色葡萄球菌、銅綠假單胞菌等的BF具有清除和破壞作用。它可使編碼金黃色葡萄球菌胞外脂多糖主要成分藻酸鹽合成的相關基因algD、algR、algU表達下調,而使抑制其合成的基因mucA表達上調,進而抑制其BF的形成;亦能夠通過抑制編碼銅綠假單胞菌密度感應(quorum sensing,QS)系統(tǒng)信號分子AHL合成酶的基因las I和rhl I的表達,使信號分子AHL的合成減少,進而抑制其BF的形成。本實驗室前期研究亦發(fā)現(xiàn)AMB對流感嗜血桿菌體外形成的BF有破壞和清除作用,且聯(lián)合抗生素的使用可大大增加抗殺菌效果。但其清除和破壞作用機制尚未闡明,亦未見相關報道。本課題從流感嗜血桿菌游離態(tài)、形成BF的早期粘附階段以及成熟的BF三個階段入手,探究AMB抗流感嗜血桿菌BF的機制。實驗選取流感嗜血桿菌標準菌株ATCC24479和臨床分離的成膜能力較強的Hi23為實驗菌株。首先,連續(xù)動態(tài)觀察不同濃度的AMB對未形成BF的浮游態(tài)的流感嗜血桿菌的作用,對比培養(yǎng)基下流感嗜血桿菌生長情況,繪制其生長曲線;其次,采用半定量實驗檢測不同濃度的AMB對流感嗜血桿菌BF形成早期的粘附情況的影響,標本經染色、脫色等處理后,以多功能酶標儀檢測其A570的OD值,并行統(tǒng)計分析;再次,體外建立流感嗜血桿菌成熟BF,以掃描電鏡鑒定其生成情況,試劑盒法提取流感嗜血桿菌成熟BF的總RNA,微量分光光度計鑒定其純度和濃度質量后行逆轉錄,再行實時熒光定量反應,比較分析不同濃度AMB作用下,流感嗜血桿菌BF形成相關基因luxS、pilA、licD的mRNA表達情況。根據(jù)不同時間流感嗜血桿菌生長情況繪制生長曲線發(fā)現(xiàn),不同濃度的AMB對ATCC24479和Hi23兩株菌浮游態(tài)生長影響較小,曲線未表現(xiàn)出明顯差異。而將培養(yǎng)了不同時間的ATCC24479和Hi23兩株菌的96孔板經結晶紫染色后以酶標儀檢測,統(tǒng)計分析A值顯示:2株6組流感嗜血桿菌經不同時間培養(yǎng)后,都有不同程度早期粘附的BF形成,與培養(yǎng)基對照組相比,不同濃度的AMB均可減少流感嗜血桿菌早期的粘附(P0.05),從而抑制其BF的進一步生成;高濃度的AMB作用更加明顯,表現(xiàn)出濃度依賴性(P0.05)。成功于體外建立流感嗜血桿菌BF模型,并完成BF總RNA的提取、逆轉錄和熒光定量PCR反應等試驗,實驗結果分析顯示:空白對照組luxS、pilA、licD基因表達最高;兩實驗組與對照組相比,luxS、pilA、licD基因表達量均明顯減少,差異具有統(tǒng)計學意義(P0.05);AMB(3.75mg/ml)組與AMB(1.875mg/ml)組相比lux S、pilA、licD基因表達均減少更明顯,差異具有統(tǒng)計學意義(P0.05)。綜上實驗得出:AMB對浮游態(tài)的流感嗜血桿菌的生長無明顯影響;AMB可減弱流感嗜血桿菌BF形成早期的粘附作用,進而影響其BF的進一步生成;AMB可下調流感嗜血桿菌BF形成相關基因luxS、pilA、licD的表達,進而影響其形成和進一步成熟。本課題為AMB在臨床的使用提供了理論基礎,可以為臨床治療流感嗜血桿菌BF感染性疾病提供有益的用藥指導。
[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.
【學位授予單位】：河北北方學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R446.5

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