【摘要】：細(xì)菌生物膜引發(fā)的慢性感染是臨床上棘手的問題之一。由于細(xì)菌生物膜對抗生素具有高度耐藥性,而在缺乏新抗生素或者高效抗菌策略的前提下,只能加大現(xiàn)有抗生素的使用劑量、延長給藥時(shí)間。長此以往,容易導(dǎo)致耐藥性和耐藥菌株的產(chǎn)生,同時(shí),抗生素的毒副作用也會(huì)增加。因此,通過改良抗生素的現(xiàn)有給藥方式,降低抗生素的使用劑量,提高作用效果,可以為細(xì)菌生物膜感染的治療提供更好的解決辦法。本文采用生物相容性好的帶正電荷的溶菌酶修飾帶負(fù)電荷的脂質(zhì)體,提高了脂質(zhì)體的穩(wěn)定性,增強(qiáng)了對細(xì)菌生物膜的親和力,提升了慶大霉素對生物膜的作用效果。具體研究內(nèi)容如下:采用1,2-棕櫚酰磷脂酰甘油(DPPG)和二棕櫚酰磷脂酰膽堿(DPPC),通過擠壓法制備了慶大霉素脂質(zhì)體(LG),經(jīng)磷鎢酸鈉沉淀法測定可知慶大霉素的包封率為0.6%。其次利用靜電作用將帶正電荷的溶菌酶吸附到負(fù)電荷的脂質(zhì)體表面得到溶菌酶修飾的慶大霉素脂質(zhì)體(LLG)。根據(jù)表面Zeta電位由LG的負(fù)值向LLG的正值轉(zhuǎn)變可知,溶菌酶成功吸附到了脂質(zhì)體表面。通過定時(shí)檢測48 h內(nèi)LG及LLG的粒徑變化可知,溶菌酶的引入可以穩(wěn)定脂質(zhì)體。利用紫外分光光度計(jì)實(shí)時(shí)監(jiān)測72 h內(nèi)LG及LLG體外釋放慶大霉素含量發(fā)現(xiàn),LG在前48 h內(nèi),慶大霉素釋放量超過了50%,而在72 h時(shí)70%以上的藥物已經(jīng)釋放。相反地,在72 h時(shí)也僅有14%的藥物從LLG中釋放出來。由此也證明了溶菌酶吸附到慶大霉素脂質(zhì)體表面可以有效改善脂質(zhì)體的穩(wěn)定性。通過體外構(gòu)建銅綠假單胞桿菌(革蘭氏陰性菌)和金黃色葡萄球菌(革蘭氏陽性菌)生物膜,利用結(jié)晶紫染色法和MTT實(shí)驗(yàn)評價(jià)發(fā)現(xiàn),與單獨(dú)慶大霉素相比,LLG在破壞已形成的細(xì)菌生物膜和抑制細(xì)菌生物膜形成時(shí)更為有效。通過熒光顯微鏡及掃描電鏡觀察細(xì)菌生物膜發(fā)現(xiàn),LLG不僅破壞了生物膜的形態(tài)及內(nèi)部結(jié)構(gòu),也影響了單個(gè)細(xì)菌的形態(tài)結(jié)構(gòu)。通過熒光定量羅丹明B與細(xì)菌生物膜的結(jié)合量,探討了LLG破壞細(xì)菌生物膜的潛在機(jī)制,結(jié)果顯示,LLG能更有效的吸附于負(fù)電荷的生物膜,進(jìn)而定點(diǎn)釋放藥物,從而達(dá)到增強(qiáng)LLG的抗生物膜效果。綜上所述,溶菌酶穩(wěn)定脂質(zhì)體的策略是可行的,LLG能夠在一定程度上降低抗生素的使用劑量,減緩耐藥菌株的形成,同時(shí),也有助于減小抗生素的毒副作用。該策略為使用抗生素治療細(xì)菌生物膜相關(guān)的頑固性感染提供了一種新的方案。
[Abstract]:Chronic infection caused by bacterial biofilm is one of the thorny problems in clinic. Because bacterial biofilms are highly resistant to antibiotics, the dosage of existing antibiotics can only be increased and the administration time can be prolonged under the premise of lack of new antibiotics or effective antibacterial strategies. In the long run, it is easy to cause drug resistance and resistant strains, and the toxic side effects of antibiotics will also increase. Therefore, by improving the current way of administration of antibiotics, reducing the dosage of antibiotics and increasing the effect of action, we can provide a better solution for the treatment of bacterial biofilm infection. In this paper, the biocompatible lysozyme with positive charge was used to modify the negatively charged liposome, which improved the stability of liposome, enhanced the affinity to bacterial biofilm, and enhanced the effect of gentamicin on biofilm. The main contents are as follows: the entrapment efficiency of gentamicin liposome (LG), was determined by sodium phosphotungstate precipitation method. The entrapment efficiency of gentamicin was 0.6 by extrusion method with 1 ~ 2-palmitylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC),). Secondly, the positive charge lysozyme was adsorbed on the surface of the negatively charged liposome by electrostatic action to obtain the lysozyme modified gentamicin liposome (LLG). According to the change of surface Zeta potential from negative value of LG to positive value of LLG, lysozyme was successfully adsorbed on the surface of liposome. The changes of LG and LLG particle size in 48 h were determined. The results showed that the introduction of lysozyme could stabilize liposomes. The release of gentamicin from LG and LLG in vitro was monitored by UV spectrophotometer in real time within 72 h. It was found that in the first 48 h, the release of gentamicin exceeded 50%, and more than 70% of the drugs had been released at 72 h. In contrast, only 14% of the drugs released from LLG at 72 h. It is also proved that lysozyme adsorbed on the surface of gentamicin liposomes can effectively improve the stability of liposomes. The biofilms of Pseudomonas aeruginosa (Gram-negative bacteria) and Staphylococcus aureus (Gram-positive bacteria) were constructed in vitro. Compared with gentamicin alone, LLG is more effective in destroying and inhibiting bacterial biofilm formation. Fluorescence microscope and scanning electron microscope (SEM) showed that LLG not only destroyed the morphology and internal structure of biofilm, but also affected the morphological structure of single bacteria. The potential mechanism of LLG destroying bacterial biofilm was discussed by fluorescence quantitative binding amount of Rhodamine B to bacterial biofilm. The results showed that LLG could be more effectively adsorbed on negative charge biofilm and then released drugs on site. In order to enhance the anti-biofilm effect of LLG. To sum up, the strategy of lysozyme stabilizing liposome is feasible. LLG can reduce the dosage of antibiotics to a certain extent, slow down the formation of drug-resistant strains, but also help to reduce the toxic side effects of antibiotics. This strategy provides a new strategy for the treatment of bacterial biofilm-related obstinate infections with antibiotics.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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