【作者】 周璐；

【导师】 李学锋； 杜凤沛；

【作者基本信息】 中国农业大学 ， 农药学， 2015， 博士

【摘要】 目前抗生素的大量使用甚至滥用导致抗药性的现象日益严重,抗菌肽因为具有杀菌作用又不易产生抗药性而备受关注。这主要因为其作用靶标为生物膜,这种独特的抗菌机理使其成为抗菌甚者抗肿瘤领域的研究热点。对于抗菌肽与模拟生物膜之间相互作用机理的研究报道很多,其中也有不少是关于两亲性的抗菌肽在脂质体表面形成孔洞的理论研究,但是对孔洞形成机理的动力学方面,尤其在实验条件下关于这一动态过程的检测与验证研究并未见报道。本论文的研究重点是以二肉豆蔻酰磷脂酰胆碱(1,2-dimyristoyl-sn-glycero-3-phophocholine, DMPC)制备的脂质体(Liposome)作为细胞膜的模型,通过调控反应温度,抗菌肽(Melittin)的浓度,脂质体的直径以及包埋在脂质体里面的荧光染料的尺寸等条件,来研究Melittin在DMPC脂质体表面形成孔洞机理的动态过程。本论文首次在实验条件下测得Melittin与DMPC脂质体作用过程中,释放染料分子前形成孔洞所需要的时间(Lag-time)及形成孔洞时Melittin的阈值浓度。通过改变实验条件的温度,定量研究Lag-time与Melittin在脂质体单位表面积的个数Q(N/nm2)之间的定量关系。荧光实验结果表明孔洞形成的过程是Melittin首先在脂质体表面形成一个临界孔洞,随着时间的延长,孔洞直径不断扩大,直到可以释放染料分子的尺寸,并且孔洞直径扩大的速率在一定范围内与Melittin的浓度有关。在荧光实验的基础上,通过圆二色谱(CD)实验,根据Melittin二级结构α-螺旋结构的百分含量变化来研究孔洞形成过程。CD实验结果表明在实验条件下发现Melittin在脂质体悬浮液中可以形成α-螺旋的二级结构。Melittin的浓度一定时,具有α-螺旋二级结构的Melittin分子的百分含量随脂质体浓度升高而增加；但是在实验条件下未发现Melittin的α-螺旋结构的百分含量与反应时间的相关性；又通过改变实验条件下的温度,发现α-螺旋的百分含量随温度的降低而减少,在一定程度上解释了荧光实验中低温下可以测得Lag-time,而在温度升高时难以测得Lag-time。本论文还设计了利用共聚焦激光扫描显微镜(Confocal laser scanning microscope,CLSM)定点扫描的方法,首次采用“微分区域法”处理成像结果,将拍摄中得到的脂质体图像在平面上微分成n(8)个圆心在同一水平直线上,半径为r(100nm)的小圆区域。通过在平面上,将直线及上面的小圆区域绕垂直与平面并过脂质体圆心O的直线旋转一周,测得该直线旋转过不同角度θ时的,每个小圆区域内荧光强度,通过绘制荧光强度随时间变化的曲线,以研究在这一过程中脂质体内荧光物质的运动规律。通过比较每个小圆区域内荧光强度变化规律,证明应用“微分区域法”测量绘制的荧光强度随区域的变化及分布图,可以确定脂质体内荧光染料的流动方向,证明在脂质体表面一定范围的位置形成孔洞,导致脂质体内的染料分子流出脂质体。本文还推导了体系的自由能变化,构建了Lag-time的计算模型,讨论了抗菌肽在脂质体表面形成孔洞的动态过程,并且通过理论计算与实验数据的基本吻合,证明了模型的可适用性。更多还原

【Abstract】 Antimicrobial peptides (AMP) represent a class of compounds to combat antibiotic resistance to microor-ganisms, neutralize biological warfare agents, and as topical antimicrobial agents. AMP kills microbialcells through insertion and permeabilization of the cytoplasmic membranes. It is important to pre-dict the efficacy of AMP at low concentration to circumvent their toxicity. Leakage of fluorescentdyes (calcein, FD4and FD20) of different molecular weights entrapped within1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/cholesterol mixed liposomes by AMP melittin indicated (i) a criticalmelittin concentration for pore formation and (ii) a lag time for pore formation above this critical con-centration. The lag time decreased with an increase in melittin concentration and was in the orderFD20> FD4> calcein.%α helix of melittin increased when exposed to liposome with this increase beingmore pronounced at higher concentrations eventually reaching an asymptotic value. The rate of dyeleakage following the lag time was found to be larger at higher melittin concentration. A simplifiedmathematical model for nucleation and growth of pores formed by an aggregate of melittin in lipidbilayer is proposed to predict the variation of rate of dye leakage with melittin concentration whichagreed fairly well with the data for calcein and seems to suggest a toroidal mechanism of pore formationwith participation of large number of phospholipid heads.By means of a new attempt of data ananlysis, Confocal microscopy images clearly indicate the disruption of the liposome by Melittin above a threshold concentration.The key of the new method is the segmentation of the fullscreen image of liposomes.The fluorescence intensity is meared of each micro-circle region and show the dye flow direction which suggest a position of the pore formation in the surface of the liposomes.Evolution of fluorescence intensity due to leakage of dyes ofdifferent sizes from liposome by melittin of different concentrations indicated a critical melittin concentration for pore formationand a lag time above this critical concentration which was largerfor lower melittin concentration as well as for larger size of dyemolecule. The rate of dye leakage following the lag time was foundto be larger at higher melittin concentration. A mathematicalmodel for nucleation and growth of pores formed by an aggregate of melittin in lipid bilayer is proposed. The difference in the lagtime for dye leakage for the three dyes of different sizes is shownto be due to the difference in the time of growth of pores from thecritical pore size to the size of the molecule.Model prediction ofvariation of dye leakage rate with melittin concentration agreedfairly well with the experimental data for calcein and seems tosuggest a toroidal mechanism of pore formation with participationof large number of phospholipid heads.更多还原