金黄色葡萄球菌噬菌体裂解酶LysGH15外用制剂的初步研制

发布时间：2018-03-31 02:06

本文选题：金黄色葡萄球菌　切入点：噬菌体裂解酶　出处：《吉林大学》2015年硕士论文

【摘要】：金黄色葡萄球菌（Staphyloccocus aureus）是人和动物表皮及呼吸道广泛存在的重要致病菌，对人和家畜健康有着巨大的危害。耐甲氧西林金黄色葡萄球菌（methicilin-resistant S. aureus, MRSA）的出现给该菌感染性疾病的临床治疗带来了极大的困难。噬菌体裂解酶（bacteriophage lysin）是噬菌体在感染宿主菌末期表达的蛋白质，它通过水解细菌细胞壁的肽聚糖使子代噬菌体释放，从而杀死细菌。因此，裂解酶很有潜力成为治疗细菌感染疾病的新型药物[1]。实验室前期获得了对金黄色葡萄球菌具有广谱、高效裂解活性的噬菌体裂解酶LysGH15，该裂解酶尤其对耐药菌株表现出高效均一的裂解活性。本研究在前期研究基础上，继续深入研制LysGH15的外用复方膏剂，确定有效的配方，并检测获得的复方膏剂治疗金黄色葡萄球菌引起的烧伤或烫伤感染模型的效果，从而为裂解酶LysGH15的外用治疗奠定基础。首先，本研究对噬菌体裂解酶LysGH15的外用膏剂进行了配方的初步确定，并对其进行了优化和改进，最终获得了复方凝胶制剂的配方：以金黄色噬菌体裂解酶LysGH15为主药，库拉索芦荟冻干粉为辅药，配以卡波姆940、甘油、氮酮、水等。进一步对LysGH15复方凝胶制剂的安全性进行了全面的评价。表明：其对小鼠皮肤不具有急性毒性，，亦不能引起过敏反应。然后，进一步利用金黄色葡萄球菌USA300建立了烧烫伤的小鼠感染模型。首先利用胶带对小鼠的皮肤进行多次粘贴，造成其皮肤黏膜的破溃，然后在创口处接种一定量的USA300菌株，发现106至108cfu的菌量能够获得金黄色葡萄球菌的烧烫伤感染模型。其创面的表皮层及真皮浅层受损，创面有明显水泡，湿润，基底潮红，并有疼痛感，水泡破裂后创面呈红白色，毛细血管扩张、出血。利用所建立的金黄色葡萄球菌引起的小鼠烧烫伤感染模型对LysGH15复方凝胶制剂的治疗效果进行了检测和评价。对其治疗结果表明应用复方凝胶制剂可以在治疗后七天就能使感染菌量下降4至5个数量级，相比非治疗组，治疗效果显著；治疗组小鼠的组织炎性细胞浸润明显减少，且创面愈合良好。表明LysGH15复方凝胶制剂在烧烫伤感染动物模型的治疗方面具有良好的效果。通过本研究成功获得了LysGH15的复方凝胶制剂，该制剂安全性良好，并且对金黄色葡萄球菌引起的小鼠烧烫伤感染模型表现出了良好的治疗效果。该研究初步确定了噬菌体裂解酶LysGH15的外用剂型和配方，为LysGH15的外部治疗应用提供了研究基础。
[Abstract]:Staphylocus aureus (Staphylocus aureus) is an important pathogen in human and animal epidermis and respiratory tract. The emergence of methicilin-resistant Staphylococcus aureus (MRSAA) has brought great difficulties to the clinical treatment of the bacterial infectious disease. Bacteriophage lysinis is a bacteriophage. Proteins expressed at the end of host bacteria, It kills bacteriophages by hydrolyzing peptidoglycan from the cell wall of bacteria. Therefore, lyase has the potential to become a new drug for the treatment of bacterial infections [1]. LysGH15, a bacteriophage lyase with high lytic activity, especially showed high and uniform lytic activity against drug-resistant strains. On the basis of previous studies, this study continued to develop the compound plaster for external use of LysGH15, and to determine the effective formula. The efficacy of compound plaster in the treatment of staphylococcus aureus burn or scalded infection model was examined, which laid a foundation for the external treatment of lyase LysGH15. Firstly, the formulation of external paste of bacteriophage lyase LysGH15 was preliminarily determined, and optimized and improved. Finally, the formula of compound gel preparation was obtained, which was mainly composed of golden yellow bacteriophage lyase LysGH15. Aloe vera lyophilized powder, supplemented with carbomer 940, glycerol, azone and water, was used to evaluate the safety of LysGH15 compound gel preparation. Also cannot cause the allergic reaction. Then, the infection model of burned mice was established by using Staphylococcus aureus USA300. First, adhesive tape was used to paste the skin of mice many times, resulting in the breaking of skin and mucous membrane, and then a certain amount of USA300 strain was inoculated at the wound. It was found that the infection model of staphylococcus aureus burn and scald could be obtained from the amount of bacteria from 106 to 108cfu. The surface of the wound was damaged in the epidermis and dermis. The wound had obvious blisters, wetness, redness to the base, and a sense of pain. The wound was red and white after the blisters burst. Capillaries dilate and bleed. The therapeutic effect of LysGH15 compound gel preparation was tested and evaluated by using the model of burn and scald infection induced by Staphylococcus aureus in mice. The results showed that the compound gel preparation could be used after treatment. Seven days can reduce the number of infected bacteria by four to five orders of magnitude. Compared with the non-treatment group, the effect of treatment was significant, the infiltration of tissue inflammatory cells in the treatment group was significantly reduced, and the wound healed well, which indicated that LysGH15 compound gel preparation had a good effect on the treatment of burned and scalded infected animal model. Through this study, the compound gel preparation of LysGH15 was successfully obtained, and the safety of the preparation was good. In addition, it showed a good therapeutic effect on the model of burn and scald infection induced by Staphylococcus aureus in mice. This study preliminarily determined the formulation and formulation of phage lyase LysGH15, which provided the basis for the external treatment of LysGH15.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S859.79

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