本文選題：金黃色葡萄球菌　切入點：噬菌體裂解酶　出處：《吉林大學(xué)》2015年碩士論文

【摘要】：金黃色葡萄球菌（Staphyloccocus aureus）是人和動物表皮及呼吸道廣泛存在的重要致病菌，對人和家畜健康有著巨大的危害。耐甲氧西林金黃色葡萄球菌（methicilin-resistant S. aureus, MRSA）的出現(xiàn)給該菌感染性疾病的臨床治療帶來了極大的困難。噬菌體裂解酶（bacteriophage lysin）是噬菌體在感染宿主菌末期表達的蛋白質(zhì)，它通過水解細菌細胞壁的肽聚糖使子代噬菌體釋放，從而殺死細菌。因此，裂解酶很有潛力成為治療細菌感染疾病的新型藥物[1]。實驗室前期獲得了對金黃色葡萄球菌具有廣譜、高效裂解活性的噬菌體裂解酶LysGH15，該裂解酶尤其對耐藥菌株表現(xiàn)出高效均一的裂解活性。本研究在前期研究基礎(chǔ)上，繼續(xù)深入研制LysGH15的外用復(fù)方膏劑，確定有效的配方，并檢測獲得的復(fù)方膏劑治療金黃色葡萄球菌引起的燒傷或燙傷感染模型的效果，從而為裂解酶LysGH15的外用治療奠定基礎(chǔ)。 首先，本研究對噬菌體裂解酶LysGH15的外用膏劑進行了配方的初步確定，并對其進行了優(yōu)化和改進，最終獲得了復(fù)方凝膠制劑的配方：以金黃色噬菌體裂解酶LysGH15為主藥，庫拉索蘆薈凍干粉為輔藥，配以卡波姆940、甘油、氮酮、水等。進一步對LysGH15復(fù)方凝膠制劑的安全性進行了全面的評價。表明：其對小鼠皮膚不具有急性毒性，，亦不能引起過敏反應(yīng)。 然后，進一步利用金黃色葡萄球菌USA300建立了燒燙傷的小鼠感染模型。首先利用膠帶對小鼠的皮膚進行多次粘貼，造成其皮膚黏膜的破潰，然后在創(chuàng)口處接種一定量的USA300菌株，發(fā)現(xiàn)106至108cfu的菌量能夠獲得金黃色葡萄球菌的燒燙傷感染模型。其創(chuàng)面的表皮層及真皮淺層受損，創(chuàng)面有明顯水泡，濕潤，基底潮紅，并有疼痛感，水泡破裂后創(chuàng)面呈紅白色，毛細血管擴張、出血。 利用所建立的金黃色葡萄球菌引起的小鼠燒燙傷感染模型對LysGH15復(fù)方凝膠制劑的治療效果進行了檢測和評價。對其治療結(jié)果表明應(yīng)用復(fù)方凝膠制劑可以在治療后七天就能使感染菌量下降4至5個數(shù)量級，相比非治療組，治療效果顯著；治療組小鼠的組織炎性細胞浸潤明顯減少，且創(chuàng)面愈合良好。表明LysGH15復(fù)方凝膠制劑在燒燙傷感染動物模型的治療方面具有良好的效果。 通過本研究成功獲得了LysGH15的復(fù)方凝膠制劑，該制劑安全性良好，并且對金黃色葡萄球菌引起的小鼠燒燙傷感染模型表現(xiàn)出了良好的治療效果。該研究初步確定了噬菌體裂解酶LysGH15的外用劑型和配方，為LysGH15的外部治療應(yīng)用提供了研究基礎(chǔ)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S859.79

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