广泛耐药鲍曼不动杆菌—秀丽隐杆线虫感染模型的构建及其在抗菌中药筛选中的应用

发布时间：2018-05-16 16:18

本文选题：广泛耐药鲍曼不动杆菌 + 秀丽隐杆线虫　；参考：《广东药科大学》2017年硕士论文

【摘要】：目的:鲍曼不动杆菌(Acinetobacter baumannii,A.baumannii)生命力强,耐药性严重,呈现世界流行趋势,已经成为我国临床最常见的致病菌之一。然而,抗生素的研发速度滞后于细菌的耐药速度,出现无药可用的局面,针对此情况,各国关于鼓励开发抗生素的优惠政策相继出台,希望通过此措施进一步加速抗生素研发的速度。因此抗生素研发不仅意义重大,也符合国际药物研发趋势。本课题拟构建广泛耐药鲍曼不动杆菌(Extensively drug-resistant Acinetobacter baumannii,XDR A.baumannii)-秀丽隐杆线虫感染模型,并利用该模型筛选清热解毒中药提取物,以期能够寻找到抗该菌的中药提取物。方法:1.选用XDR A.baumannii临床菌株GZLC-1作为致病菌,glp-4(bn2ts);sek-1(km4)秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)作为模型动物,构建广泛耐药鲍曼不动杆菌-秀丽隐杆线虫感染模型。通过比较线虫在M9缓冲液、10%BHI-M9、20%BHI-M9、BHI等几种液体培养基中存活时间,确定最适比例的液体培养基。为了增强A.baumannii的致病性,进一步优化了铁离子在液体培养基的加入量。最后确定该感染模型液体培养基的组成成分。2.比较不同感染时长下线虫的存活时间,确定线虫的最佳感染时长。在最佳感染时长下进一步确定最佳感染菌浓度和治疗时长。3.分别用水,50%乙醇,95%乙醇三种溶剂对28味清热解毒中药进行回流提取,药液比是1:10,提取两次,每次一小时,提取液合并,放在水浴锅上干燥后制成粉末后放置4℃冰箱待用。4.采用广泛耐药鲍曼不动杆菌-线虫感染模型对中药提取物进行筛选,每种提取物配成1000μg/ml,100μg/ml,10μg/ml,1μg/ml,0.1μg/ml五个浓度进行药效筛选实验,同时设阴性对照(1%DMSO)和阳性对照(1%DMSO溶解2μg/ml多粘菌素B)。5.选取体内抗A.baumannii效果较好的中药提取物进行体外药敏实验,比较中药提取物在体内与体外的抗A.baumannii效果。结果:1、广泛耐药鲍曼不动杆菌-秀丽隐杆线虫感染模型培养基组成成分是10%BHI-M9、5μg/ml萘啶酸、10μM Fe Cl3、1%DMSO。2、广泛耐药鲍曼不动杆菌-秀丽隐杆线虫感染模型感染时长是6h,感染菌浓度是5×105CFU/ml,治疗时长是24h。3、线虫体内抗A.baumannii效果较好的提取物(与阴性对照组相比存活率提高到4.5倍以上)有黄芩、大青叶、牡丹皮、鸡骨草、何首乌、山豆根等六味中药的水提取物。4、黄芩、大青叶、牡丹皮、鸡骨草、何首乌、山豆根等六味中药的水提取物对XDR A.baumannii GZLC-1的MIC值皆大于2048μg/ml。结论:1、本实验利用临床XDR A.baumannii GZLC-1,建立了广泛耐药鲍曼不动杆菌-线虫感染模型,并证实线虫的死亡是由于XDR A.baumannii在线虫体内定殖且不断繁殖导致。2、黄芩、大青叶、牡丹皮、鸡骨草、何首乌、山豆根等六种中药水提取物对感染了XDR A.baumannii的线虫具有较好的治疗作用。3、黄芩、大青叶、牡丹皮、鸡骨草、何首乌、山豆根等六种中药水提取物体外抗XDR A.baumannii GZLC-1的效果较差,但体内药效结果较好,说明这六种提取物可能不是直接杀菌,而是从提高免疫力等其他途径提高抗菌效果。
[Abstract]:Objective: Acinetobacter baumannii (Acinetobacter, A.baumannii), which has strong vitality and serious resistance, presents a worldwide trend and has become one of the most common pathogenic bacteria in our country. However, the speed of research and development of antibiotics lags behind the rate of antibiotic resistance, and there is a situation of no drug availability. The preferential policies of antibiotics have been introduced in succession, hoping to further accelerate the speed of antibiotic research and development. Therefore, the research and development of antibiotics is not only significant, but also in line with the trend of international drug development. This topic is to construct Extensively drug-resistant Acinetobacter baumannii (XDR A.baumannii) - the beautiful hidden secret The model of nematode infection and the use of this model to screen the extract of Chinese herbal medicine for clearing heat and detoxification in order to find the extract of Chinese traditional medicine. Method: 1. XDR A.baumannii clinical strain GZLC-1 was selected as pathogenic bacteria, glp-4 (bn2ts); SEK-1 (km4) Caenorhabditis elegans (Caenorhabditis elegans, C.elegans) was used as model animal to construct extensive drug resistance. The infection model of Acinetobacter Bauman, Caenorhabditis elegans, determines the optimum proportion of liquid medium by comparing the survival time of nematodes in several liquid medium such as M9 buffer, 10%BHI-M9,20%BHI-M9, BHI and other liquid medium. In order to enhance the pathogenicity of A.baumannii, the addition of iron ions in liquid medium is further optimized. Finally, the infection is determined. The composition of the model liquid medium,.2., compared the survival time of the long line worm in different infections, and determined the best time for the nematode infection. At the time of the best infection, the optimum concentration of the infected bacteria and the treatment time of.3., 50% ethanol, and 95% ethanol, respectively, were reflued and extracted by three kinds of solvents. It is at 1:10, two times, two times, one hour each time, the extraction solution is merged, then dried in a water bath and then made into powder and then placed at 4 degrees centigrade. The.4. Acinetobacter - Acinetobacter - nematode infection model is selected to select the extract of traditional Chinese medicine. Each extract is matched with 1000 mu g/ml, 100 mu g/ml, 10 mu g/ml, 1 mu g/ml, 0.1 u g/ml five concentration. Screening test, simultaneously setting negative control (1%DMSO) and positive control (1%DMSO dissolving 2 g/ml polymyxin B).5. to select the in vivo anti A.baumannii effect of traditional Chinese medicine extracts in vitro drug sensitivity test, compare the anti A.baumannii effect of traditional Chinese medicine extracts in vivo and in vitro. Fruit: 1, widely resistant Acinetobacter Bauman - Caenorhabditis elegans sense The composition of the culture medium was 10%BHI-M9,5 mu g/ml naphthalene acid, 10 mu M Fe Cl3,1%DMSO.2, the widespread resistance of the widely resistant Acinetobacter spp. - Caenorhabditis elegans infection model was 6h, the concentration of infected bacteria was 5 * 105CFU/ml, the treatment time was 24h.3, and the anti A.baumannii effect of nematode was better than that of the negative control group. Up to 4.5 times more than 4.5 times) the water extracts of six Chinese herbs,.4, Scutellaria, peony, peony, peony, peony, Radix Polygoni Multiflori, pea root and other six Chinese herbs, are all more than 2048 mu g/ml. conclusion: 1. 1, this experiment uses clinical XDR A.baumannii. GZLC-1, a widely drug resistant Acinetobacter Bauman nematode infection model was established, and the death of the solid line worm was due to the colonization of XDR A.baumannii in the worm and the continuous propagation of.2, Scutellaria, peony skin, chicken bone grass, Polygonum multiflorum, pea root and so on, which had good treatment for the XDR A.baumannii infected nematodes. The effects of.3, Scutellaria, Radix Scutellariae, pony leaf, peony skin, chicken bone grass, Radix Polygonum multiflorum, Radix Sophorae root and other six kinds of aqueous extracts were less effective against XDR A.baumannii GZLC-1, but the results were good, indicating that these six kinds of extracts may not be directly germicidal, but improve the antibacterial effect from other ways.

【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332;R285

【参考文献】