钾、钠和钙离子对两性霉素B与脂膜相互作用的影响

发布时间：2018-06-25 21:06

本文选题：两性霉素B + 膜孔洞　；参考：《陕西师范大学》2016年博士论文

【摘要】：两性霉素B是一种广谱性抗真菌多烯类药物,它与真菌细胞膜的麦角甾醇结合,在膜上形成孔洞,使得细胞内重要的生命物质外漏,有毒物质内渗,从而使真菌生命力下降甚至死亡,由此达到杀真菌的作用。而两性霉素B与哺乳动物细胞膜上存在的甾醇(胆固醇)分子也容易结合,破坏细胞膜,从而产生毒副作用。这种毒副作用非常严重,特别是肾毒性,长期使用该药物还会导致肾及循环系统的损害,表现为蛋白尿、氮质血症、低血钾、贫血等,故极大地限制了两性霉素B在临床上的应用。虽然,近年来已将两性霉素B制成脂质体药剂应用于临床治疗,但其对宿主细胞的毒性仍然很大。因此,两性霉素B的分子作用机制以及其细胞毒性机理已成为一个重要的科学问题。两性霉素B的抗真菌活性依赖于细胞膜上的甾醇类物质,其与甾醇类物质在细胞内膜形成孔洞(或者离子通道)。这种孔洞影响正常的生理离子运输,特别是钾离子的运输。两性霉素B的这种离子通道活性不仅与甾醇有关,也和其它膜成分间的相互作用有关。与不饱和磷脂相比,两性霉素B与饱和磷脂的相互作用更强。有些学者认为两性霉素B的抗真菌活性与其在细胞膜上的聚集有关,而且钙离子对其聚集程度产生一定的影响。高钠盐的摄入可能降低两性霉素B对哺乳动物细胞的毒性。本课题从饱和磷脂DPPC膜与含有胆固醇的DPPC膜两大膜体系入手,研究两性霉素B在钾、钠或钙金属离子环境下与膜的相互作用。不仅研究了两性霉素B在含有或者不含胆固醇的膜上的活性有何不同,而且也研究了不同的单一金属阳离子对它与这两种膜相互作用的影响,旨在揭示两性霉素B对哺乳动物细胞膜毒性的机理,以及钾、钠、钙离子对其细胞毒性有何影响,这对降低细胞毒性,扩展该药物的应用具有一定的意义。第二章和第三章主要利用Langmuir单层膜模型系统从热力学角度研究了不同金属阳离子对两性霉素B与DPPC界面膜及Chol/DPPC界面膜的相互作用。通过表面压力-单分子面积曲线计算出体系的过量吉布斯自由能、焓变和熵变以及弹性模量,结果表明:(1)不同的金属阳离子对不同比例的AmB/DPPC混合单层膜的相变产生影响。当X_(AmB)=0.7时,与无金属离子情况相比,钙离子的存在使AmB/DPPC混合单层膜的相变处表面压力和相应的弹性模量明显增大,这与钾离子或钠离子存在时的现象明显不同。钾离子或钠离子存在时,与无金属离子相比,相变处表面压力减小,相应弹性模量稍微增大。不同金属离子存在时,混合单层膜发生相变时的膜成分AmB和DPPC摩尔比例不同。AmB/DPPC混合单层膜发生相变所需两性霉素B的摩尔比例在不同金属离子环境中由大到小依次为钠离子水钾离子钙离子环境。(2)无AmB时,Chol/DPPC二元混合单层脂膜处于液相-凝胶相共存态,而系统中加入AmB后,AmB/Chol/DPPC三元混合单层膜处于液态,并发生相转移,系统中出现液态凝聚相。因此,AmB的加入,可以诱导Chol/DPPC脂单层膜物理相态的变化,使凝胶相消失,出现液态凝聚相。(3)过量吉布斯自由能和二维位力物态方程理论系数分析知,Chol/DPPC二元混合单层膜和AmB/Chol/DPPC三元混合单层膜体系中分子间的相互作用力都主要表现为引力。但是AmB的加入,使单层膜体系中分子间的相互作用力减弱。并且与无金属离子相比,各金属离子的加入也使得混合膜体系中的分子间相互作用力减弱,而且这种作用力削弱程度因金属离子种类的不同而不同。另外,脂质体双层膜模型已经被广泛地应用于不同的细胞生物化学和生物物理研究,其最大优势在于脂质体可以包含各种不同的膜活性物质进入脂双层,形成人工膜结构。脂质体模型膜系统为理解生物系统的复杂生命活动提供了一个有效的研究方法。因此,第四章和第五章利用脂质体人工双层膜模型系统,用NBD基团分别位于亲水头部和疏水尾部的荧光脂探针NBD-PE和6-NBD-PC标记AmB/DPPC脂质体,通过红移效应来研究探针所处膜微环境的变化,通过稳态荧光和瞬态荧光来研究不同金属离子对荧光探针在脂质体双层膜微环境中荧光偏振及荧光寿命的影响,来探究AmB/DPPC双层膜的亲水膜区域和疏水膜区域受不同金属离子的影响,结果表明:(1)比较有和无胆固醇两种脂质体体系,钠离子环境下,胆固醇加入后,亲水和疏水区域有序性均降低,钙离子环境下恰恰相反,胆固醇加入后,亲水和疏水区域有序性均增大。而钾离子环境下,胆固醇的加入,使脂质体膜亲水区域有序性增大,疏水区域有序性减小。对比有和无胆固醇脂质体体系中荧光脂的REES值,可知加入胆固醇后,不同条件下均降低,说明胆固醇的加入使脂质体中疏水区酰基链的侧向包裹能力增大。(2)从某种角度讲,这些金属离子对胆固醇的作用产生了一定的影响。在AmB/Chol/DPPC脂质体膜体系中,胆固醇分子与两性霉素B分子形成复合物,与膜上其他分子相互作用。胆固醇诱导NBD基团向更深区域定位,说明胆固醇增强了膜的包裹能力。这一现象同时受到钾、钠和钙离子的影响。对于胆固醇对膜亲水区域的影响,钾离子无明显作用,钠离子略有抑制作用,而钙离子起促进作用。然而,对于胆固醇对疏水区域的影响,钾离子起到抑制作用,而钠离子和钙离子均起到促进作用。已有报道说明高钠盐的摄入可能改善AmB对病人的细胞毒性,这也许与钠离子对胆固醇增强膜包裹能力的促进作用相关。(3)这些金属离子对胆固醇作用的影响也许与其在膜上的离子运输方式相关。胆固醇与AmB形成的孔洞具有高钾离子通透性,胆固醇存在时,钾离子主动通过膜进入脂质体内部。但是,钠离子和钙离子通过被动运输由高浓度向低浓度穿过膜进入脂质体内部。并且,钙离子具有维持生物膜稳定性的特殊作用。两性霉素B药物进入人体后,与细胞膜上胆固醇结合形成孔洞,同时受到细胞内不同生命金属离子的不同影响,这可能与其离子运输方式不同相关。
[Abstract]:Amphotericin B is a broad-spectrum antifungal polyene drug, which combines with ergosterol in the fungal cell membrane to form holes on the membrane, making the vital substances in the cell leaking and inactivating the toxic substances, thus causing the fungal vitality to decline and even die, and thus achieve the effect of the fungicidal activity. Amphotericin B and mammalian cell membrane The existing sterol (cholesterol) molecules also easily combine to destroy the cell membrane and thus produce toxic and side effects. This toxic side effect is very serious, especially kidney toxicity. Long term use of the drug can also cause damage to the kidney and circulation system, which is characterized by proteinuria, azaemia, hypokalemia, anemia and so on, so it greatly restricts the clinic of amphotericin B in clinic. Although the use of amphotericin B into liposomes has been applied to clinical treatment in recent years, the toxicity of amphotericin B is still very large. Therefore, the molecular mechanism of amphotericin and its cytotoxic mechanism have become an important scientific problem. The antifungal activity of amphotericin B depends on the steroids on the cell membrane. An alcohol substance, which forms holes (or ion channels) with sterols in the cell lining. This hole affects normal physiological ion transport, especially the transport of potassium ions. The ionic channel activity of amphotericin B is related not only to sterols but also to the interaction between other membrane components. The interaction between the B and the saturated phospholipid is stronger. Some scholars believe that the antifungal activity of amphotericin B is related to the aggregation on the cell membrane, and the calcium ion has a certain effect on its aggregation. The intake of high sodium salt may reduce the toxicity of amphotericin B to mammalian cells. This subject is from the saturated phospholipid DPPC membrane and contains the bile. The interaction between amphotericin B and membrane in the environment of potassium, sodium or calcium metal ions was studied with the two large membrane system of sterol DPPC membrane. It not only studied the difference in the activity of amphotericin B on the membrane containing or without cholesterol, but also studied the effects of different single metal ions on the interaction between them and the two kinds of membranes. The mechanism of the toxicity of amphotericin B to mammalian cell membrane, and the effect of potassium, sodium, and calcium ion on its cytotoxicity are revealed. It is of certain significance to reduce cytotoxicity and expand the application of the drug. The second and third chapters mainly study the different metal cations from the thermodynamic point of view using the Langmuir monolayer model system. The interaction between amphotericin B and DPPC boundary mask and Chol/DPPC boundary mask. Through the surface pressure single molecule area curve, the excess Gibbs free energy, enthalpy and entropy and modulus of elasticity are calculated. The results show that: (1) different metal cations have influence on the phase transition of AmB/DPPC mixed monolayer with different proportions. When X_ (AmB) =0.7, compared with the non metal ions, the presence of calcium ions significantly increases the surface pressure and the corresponding elastic modulus at the phase transition of the AmB/DPPC mixed monolayer, which is obviously different from the presence of potassium ions or sodium ions. When the potassium ion or sodium ion exists, the surface pressure of the phase transition is reduced and the corresponding elasticity is compared with the non metal ions. When different metal ions exist, the proportion of AmB and DPPC mole in the phase transition of the mixed monolayer is different from that of.AmB/DPPC mixed monolayer. The molar ratio of amphotericin B is in the different metal ion environment from the sodium ion to potassium ion calcium ion environment. (2) Chol/DPPC without AmB. The mixed monolayer of two elements is in the coexistence state of liquid gel phase, and after adding AmB in the system, the AmB/Chol/DPPC three element mixed monolayer is in liquid, and the phase transfer occurs, and the liquid condensate phase appears in the system. Therefore, the addition of AmB can induce the change of the physical phase state of the Chol/DPPC lipid monolayer, and make the gel phase disappear and appear liquid condensed phase. (3) The analysis of the theoretical coefficient of the excess Gibbs free energy and the two-dimensional potential state equation shows that the intermolecular interaction between the Chol/DPPC two element mixed monolayer and the AmB/Chol/DPPC three element monolayer is mainly expressed as gravitational force. But the addition of AmB makes the interaction between molecules in the monolayer system weaken. And it is also associated with the non metal ions. In contrast, the addition of metal ions also reduces the intermolecular interaction in the mixed membrane system, and the degree of weakening of this force varies depending on the variety of metal ions. In addition, the liposome double layer membrane model has been widely used in different cell biology and Biophysics studies, with the greatest advantage in lipid. The body can contain a variety of different membrane active substances into the lipid bilayer and form an artificial membrane structure. The liposome model membrane system provides an effective method for understanding the complex life activities of the biological system. Therefore, the fourth and fifth chapters use the liposome artificial bilayer model system with the NBD group in the hydrophilic head and sparsely. The fluorescent probe NBD-PE and 6-NBD-PC labeled AmB/DPPC liposomes at the end of the water were used to study the changes in the microenvironment of the membrane by the red shift effect. The effects of different metal ions on the fluorescence polarization and fluorescence lifetime of the fluorescent probes in the liposome double layer microenvironment were investigated by the red shift effect. To explore the AmB/DPPC bilayer The hydrophilic membrane region and the hydrophobic membrane region are affected by different metal ions. The results are as follows: (1) there are two kinds of liposome system with and without cholesterol. Under the sodium ion environment, the order of hydrophilic and hydrophobic regions decreases after the addition of cholesterol. The order of hydrophilic and hydrophobic regions increases after the calcium ion environment is exactly opposite. In the environment of potassium ion, the addition of cholesterol increases the order of the hydrophilic region of the liposome membrane, and reduces the order of the hydrophobic region. The REES value of the fluorescent lipid in the cholesterol free liposome system is compared with that of the cholesterol free liposomes. It is known that after the addition of cholesterol, it is reduced under different conditions, indicating the addition of cholesterol to the lateral parcel of the acyl chain in the hydrophobic region of the liposome. Ability to increase. (2) in a way, these metal ions have a certain effect on the role of cholesterol. In the AmB/Chol/DPPC liposome membrane system, the cholesterol molecules and amphotericin B molecules form a complex, interacting with other molecules on the membrane. Cholesterol induces the NBD group to move deeper into the region, indicating that cholesterol enhances the membrane. This phenomenon is influenced by the effects of potassium, sodium and calcium ions. For the effect of cholesterol on the hydrophilic region of the membrane, there is no obvious effect on the potassium ion, the sodium ion has a slight inhibition, and the calcium ion promotes the effect. However, the effect of the potassium ion on the hydrophobic area is inhibited, and the sodium and calcium ions are both played. It has been reported that high sodium intake may improve the cytotoxicity of AmB to patients, which may be related to the promotion of sodium ions on the ability of cholesterol enhanced membrane encapsulation. (3) the effects of these metal ions on cholesterol may be related to the mode of ion transport on the membrane. The pores formed by cholesterol and AmB In the presence of high potassium ion permeability, when the cholesterol exists, the potassium ions enter the liposomes actively through the membrane. However, the sodium and calcium ions enter the liposomes through the passive transport from the high concentration to the low concentration through the membrane. And the calcium ion has a special role in maintaining the stability of the biofilm. Amphotericin B drugs enter the body and the cells. The cholesterol binding in the membrane forms holes and is affected by different metal ions in the cells. This may be related to the different modes of ion transport.
【学位授予单位】：陕西师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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