基于“鼻五度辨证”理论研究清胆泄热法对鼻—鼻窦表皮葡萄球菌生物膜的体外抑制作用

发布时间：2018-06-15 17:02

本文选题：鼻五度辨证 + 清胆泄热法　；参考：《成都中医药大学》2016年硕士论文

【摘要】：目的：采集“胆腑郁热”型鼻渊患者及正常人窦口鼻道复合体处分泌物,分析二者鼻-鼻窦优势需氧菌的异同。观察表皮葡萄球菌临床分离株在实验室环境下的生物膜形成能力、生长状态及生长量。人工培养表皮葡萄球菌生物膜,观察以清胆泄热法为立方依据的中成药鼻渊舒口服液对表皮葡萄球菌及其形成的生物膜的抑制作用,利用激光共聚焦显微镜观察生物膜的形态变化。基于“鼻五度辨证”理论,从鼻-鼻窦条件致病菌形成生物膜的角度进一步研究以“清胆泄热法”为立方依据的鼻渊舒口服液治疗CRS的效应机制,以期为“鼻五度辨证”理论的进一步研究提供更多的依据及研究思路。方法：1.采集“胆腑郁热”型鼻渊患者及正常人窦口鼻道复合体处分泌物,进行需氧菌常规培养,分离、鉴定优势菌。2.定性、定量表皮葡萄球菌产膜能力及生长量变化,筛选出PIA阳性表达株。3.检测鼻渊舒口服液对表皮葡萄球菌的最小抑制菌浓度以及不同浓度鼻渊舒口服液对不同时期生物膜的抑制作用。4.体外培养表皮葡萄球菌生物膜,通过激光共聚焦荧光显微镜动态观察鼻渊舒口服液抑制前后生物膜形态结构的变化。结果：1.21例样本中,9例优势菌为表皮葡萄球菌,约占需氧菌总数的42.9%。具有产膜能力的有6株,约占表皮葡萄球菌总数的66.7%,其中鼻窦炎组5株,正常组1株。2.表皮葡萄球菌游离菌含量从2h起逐渐升高,8h后出现下降趋势。PIA+组黏附菌在4h后出现,PIA-组黏附菌8h时开始增长。PIA+组、PIA-组与空白对照组均有显著差异性(P0.05)。3.鼻渊舒口服液对鼻-鼻窦表皮葡萄球菌的体外最小抑菌浓度为64倍稀释浓度。不同时间段,2倍和64倍稀浓度的抑制作用有显著差异性(P0.01)。4.激光共聚焦下见：1d时,大量细菌黏附,部分有聚拢趋势。3d时,可见膜状物及通道样改变,膜结构较松散。7d时,为整齐的三维立体膜状结构,菌团被包裹在其中,膜结构整齐致密。经鼻渊舒口服液作用后的成熟生物膜,随着抑菌时间增加,三维膜状结构遭到破坏,并逐渐瓦解、分化。结论：1.表皮葡萄球菌是OMC处分离最多的需氧菌,在CRS中产膜能力较高。在判断产膜能力上,定性和定量试验无明显差异性。2.黏附是表皮葡萄球菌形成生物膜的开始过程,并且4-8h间是形成生物膜的关键时期,此时表皮葡萄球菌生物膜进入成熟及维护期,大量游离菌被包裹其中,形成稳定的膜结构。3.在体外,64倍稀释浓度的鼻渊舒口服液即可抑制表皮葡萄球菌的黏附,随着浓度升高,抑制作用逐渐增强,当达到4倍稀释浓度时,抑制作用不再发生明显变化。4.鼻渊舒口服液对鼻-鼻窦表皮葡萄球菌生物膜的体外抑制作用可能表现为影响细菌的黏附,以及对细菌产生的多糖黏附素进行分解。
[Abstract]:Objective: to collect the secretion of sinoorhinal complex in patients with bilirubin and heat, and to analyze the similarities and differences of the dominant aerobic bacteria in the nasal and paranasal sinuses. To observe the biofilm forming ability, growth state and growth of Staphylococcus epidermidis clinical isolates in laboratory environment. The biofilm of Staphylococcus epidermidis was cultured artificially. The inhibitory effect of Biyuanshu oral liquid, a traditional Chinese patent medicine, on Staphylococcus epidermidis and its biofilm formation was observed. The morphological changes of biofilm were observed by laser confocal microscope. Based on the theory of "nasal fifth degree differentiation", the effect mechanism of Biyuanshu oral liquid on CRS was further studied from the perspective of biofilm formation of nasal and paranasal sinus conditional pathogens. In order to provide more basis and research ideas for the further study of the theory of syndrome differentiation of the fifth degree of nose. Method 1: 1. The secretions from sinoorhinal complex of patients with "gallbladder and heat" type and normal subjects were collected, and aerobic bacteria were cultured routinely, isolated and identified as dominant bacteria. Qualitative and quantitative changes of membrane production ability and growth of Staphylococcus epidermidis were identified and PIA positive expression strain. 3. 3. The minimal inhibitory bacteria concentration of Biyuanshu oral liquid on Staphylococcus epidermidis and the inhibitory effect of different concentration of Biyuanshu oral liquid on biofilm in different period were determined. Staphylococcus epidermidis biofilm was cultured in vitro. The morphology and structure of biofilm were observed by laser confocal fluorescence microscope before and after the inhibition of Biyuanshu oral liquid. Results Staphylococcus epidermidis accounted for 42.9% of the total number of aerobic bacteria. There were 6 strains with membrane producing ability, which accounted for 66.7% of the total number of Staphylococcus epidermidis, including 5 strains in sinusitis group and 1 strain in normal group. The content of free bacteria in Staphylococcus epidermidis increased gradually from 2 h to 8 h, and then decreased. The adhesion bacteria of PIA group began to increase at 8 h after the emergence of PIA-group. There was significant difference between PIA group and control group. The minimal inhibitory concentration of Biyuanshu oral liquid on Staphylococcus epidermidis in vitro was 64 times dilution concentration. The inhibitory effects of 2 times and 64 times of dilute concentration in different time periods were significantly different (P 0.01. 4). At 1: 1 d, a large number of bacteria adhered, some of them showed the change of membrane and channel at 3 d, the membrane structure was loose for 7 d, and it was a three dimensional membrane structure, in which the bacteria mass was wrapped in it. The membrane structure is neat and compact. The mature biofilm treated by Biyuanshu oral liquid, with the increase of bacteriostasis time, the three-dimensional membrane structure was destroyed, and gradually disintegrated and differentiated. Conclusion 1. Staphylococcus epidermidis is the most aerobic bacteria isolated from OMC and has higher membrane production ability in CRS. There was no significant difference between qualitative and quantitative tests in judging the ability of film production. Adhesion is the beginning process of biofilm formation of Staphylococcus epidermidis and the key period of biofilm formation is 4-8 h. When Staphylococcus epidermidis biofilm enters maturity and maintenance period, a large number of free bacteria are encapsulated in the biofilm, forming a stable membrane structure. Biyuanshu oral liquid with 64 times dilution concentration in vitro could inhibit the adhesion of Staphylococcus epidermidis. With the increase of concentration, the inhibitory effect was gradually enhanced. When the concentration reached 4 times, the inhibitory effect was no longer significantly changed .4. The inhibitory effect of Biyuanshu oral liquid on the biofilm of Staphylococcus epidermidis in vitro may be as follows: the adhesion of bacteria and the decomposition of polysaccharide adhesion produced by bacteria.
【学位授予单位】：成都中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R276.1

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