羧基荧光素脂质体的制备及其与病原菌的响应行为

Preparation of carboxyfluorescein liposomes and their response to pathogenic bacteria

【背景】由于目前临床上检测细菌感染的方法既耗时又昂贵,所以开发快速简便的鉴别方法势在必行。【目的】通过在脂质体膜材料中添加不同的稳定剂,研究它们对于脂质体稳定性的影响,并探究荧光素脂质体与病原菌之间的响应情况。【方法】以磷脂酰胆碱和胆固醇为主要原料,1,2-棕榈酰磷脂酰甘油[1,2-Dipalmitoyl-sn-glycero-3-phospho-(1?-rac-glycerol),DPPG]、十八胺和10,12-二十三二炔酸(10,12-Tricosadiyonic acid,TCDA)为稳定剂,采用薄膜分散-超声法制备羧基荧光素脂质体。利用病原菌能够分泌毒力因子造成脂质体渗漏的原理,将病原菌菌液和上清液分别与荧光素脂质体孵育,检测从脂质体渗漏的羧基荧光素的荧光强度,反映病原菌的毒性程度。【结果】DPPG和TCDA都能增加脂质体的稳定性,而十八胺的添加则导致脂质体稳定性的降低。与金黄色葡萄球菌ATCC25923和铜绿假单胞菌PAO1菌液和上清液共同孵育的脂质体荧光强度大量增加,与大肠杆菌DH5α和PBS缓冲液共同孵育的脂质体荧光强度几乎不变。【结论】能够分泌外毒素的金黄色葡萄球菌和铜绿假单胞菌都能对脂质体产生响应,而不分泌外毒素的大肠杆菌则不会对脂质体产生响应。

[Background] According to the clinical detection of wound infection is time-consuming and expensive, fast and convenient identification is imperative. [Objective] We investigated the stability of the liposomes by adding different components to the liposomal membranes, and their response to bacterial. [Methods] Liposome encapsulated carboxyfluorescein were prepared with phosphocholine（PC） as a main lipid constituent, cholesterol, 10,12-tricosadiyonic acid（TCDA）, 1,2-dipalmitoyl-sn-glycero-3-phospho-（1？-rac-glycerol）（DPPG）, octadecylamine as stabilizing agents by thin-film and rehydration method. The bacterial suspensions and respective supernatants were incubated with fluorescent liposome to obtain the fluorescence increments according to the interaction between toxins and liposomes. [Results] DPPG and TCDA increased the stability of the liposome, and octadecylamine decreased it. When incubated with bacterial suspensions and its supernatants of Staphylococcus aureus ATCC25923 and Pseudomonas aeruginosa PAO1, the obvious increase of the fluorescence intensity was observed, while incubated with Escherichia coli DH5α and PBS, the fluorescence intensity almost unchanged. [Conclusion] S. aureus and P. aeruginosa which release exotoxins can lyse liposomes but not by E. coli.