本文選題：兩性霉素B + 膜孔洞　； 參考：《陜西師范大學》2016年博士論文

【摘要】：兩性霉素B是一種廣譜性抗真菌多烯類藥物,它與真菌細胞膜的麥角甾醇結(jié)合,在膜上形成孔洞,使得細胞內(nèi)重要的生命物質(zhì)外漏,有毒物質(zhì)內(nèi)滲,從而使真菌生命力下降甚至死亡,由此達到殺真菌的作用。而兩性霉素B與哺乳動物細胞膜上存在的甾醇(膽固醇)分子也容易結(jié)合,破壞細胞膜,從而產(chǎn)生毒副作用。這種毒副作用非常嚴重,特別是腎毒性,長期使用該藥物還會導致腎及循環(huán)系統(tǒng)的損害,表現(xiàn)為蛋白尿、氮質(zhì)血癥、低血鉀、貧血等,故極大地限制了兩性霉素B在臨床上的應用。雖然,近年來已將兩性霉素B制成脂質(zhì)體藥劑應用于臨床治療,但其對宿主細胞的毒性仍然很大。因此,兩性霉素B的分子作用機制以及其細胞毒性機理已成為一個重要的科學問題。兩性霉素B的抗真菌活性依賴于細胞膜上的甾醇類物質(zhì),其與甾醇類物質(zhì)在細胞內(nèi)膜形成孔洞(或者離子通道)。這種孔洞影響正常的生理離子運輸,特別是鉀離子的運輸。兩性霉素B的這種離子通道活性不僅與甾醇有關(guān),也和其它膜成分間的相互作用有關(guān)。與不飽和磷脂相比,兩性霉素B與飽和磷脂的相互作用更強。有些學者認為兩性霉素B的抗真菌活性與其在細胞膜上的聚集有關(guān),而且鈣離子對其聚集程度產(chǎn)生一定的影響。高鈉鹽的攝入可能降低兩性霉素B對哺乳動物細胞的毒性。本課題從飽和磷脂DPPC膜與含有膽固醇的DPPC膜兩大膜體系入手,研究兩性霉素B在鉀、鈉或鈣金屬離子環(huán)境下與膜的相互作用。不僅研究了兩性霉素B在含有或者不含膽固醇的膜上的活性有何不同,而且也研究了不同的單一金屬陽離子對它與這兩種膜相互作用的影響,旨在揭示兩性霉素B對哺乳動物細胞膜毒性的機理,以及鉀、鈉、鈣離子對其細胞毒性有何影響,這對降低細胞毒性,擴展該藥物的應用具有一定的意義。第二章和第三章主要利用Langmuir單層膜模型系統(tǒng)從熱力學角度研究了不同金屬陽離子對兩性霉素B與DPPC界面膜及Chol/DPPC界面膜的相互作用。通過表面壓力-單分子面積曲線計算出體系的過量吉布斯自由能、焓變和熵變以及彈性模量,結(jié)果表明:(1)不同的金屬陽離子對不同比例的AmB/DPPC混合單層膜的相變產(chǎn)生影響。當X_(AmB)=0.7時,與無金屬離子情況相比,鈣離子的存在使AmB/DPPC混合單層膜的相變處表面壓力和相應的彈性模量明顯增大,這與鉀離子或鈉離子存在時的現(xiàn)象明顯不同。鉀離子或鈉離子存在時,與無金屬離子相比,相變處表面壓力減小,相應彈性模量稍微增大。不同金屬離子存在時,混合單層膜發(fā)生相變時的膜成分AmB和DPPC摩爾比例不同。AmB/DPPC混合單層膜發(fā)生相變所需兩性霉素B的摩爾比例在不同金屬離子環(huán)境中由大到小依次為鈉離子水鉀離子鈣離子環(huán)境。(2)無AmB時,Chol/DPPC二元混合單層脂膜處于液相-凝膠相共存態(tài),而系統(tǒng)中加入AmB后,AmB/Chol/DPPC三元混合單層膜處于液態(tài),并發(fā)生相轉(zhuǎn)移,系統(tǒng)中出現(xiàn)液態(tài)凝聚相。因此,AmB的加入,可以誘導Chol/DPPC脂單層膜物理相態(tài)的變化,使凝膠相消失,出現(xiàn)液態(tài)凝聚相。(3)過量吉布斯自由能和二維位力物態(tài)方程理論系數(shù)分析知,Chol/DPPC二元混合單層膜和AmB/Chol/DPPC三元混合單層膜體系中分子間的相互作用力都主要表現(xiàn)為引力。但是AmB的加入,使單層膜體系中分子間的相互作用力減弱。并且與無金屬離子相比,各金屬離子的加入也使得混合膜體系中的分子間相互作用力減弱,而且這種作用力削弱程度因金屬離子種類的不同而不同。另外,脂質(zhì)體雙層膜模型已經(jīng)被廣泛地應用于不同的細胞生物化學和生物物理研究,其最大優(yōu)勢在于脂質(zhì)體可以包含各種不同的膜活性物質(zhì)進入脂雙層,形成人工膜結(jié)構(gòu)。脂質(zhì)體模型膜系統(tǒng)為理解生物系統(tǒng)的復雜生命活動提供了一個有效的研究方法。因此,第四章和第五章利用脂質(zhì)體人工雙層膜模型系統(tǒng),用NBD基團分別位于親水頭部和疏水尾部的熒光脂探針NBD-PE和6-NBD-PC標記AmB/DPPC脂質(zhì)體,通過紅移效應來研究探針所處膜微環(huán)境的變化,通過穩(wěn)態(tài)熒光和瞬態(tài)熒光來研究不同金屬離子對熒光探針在脂質(zhì)體雙層膜微環(huán)境中熒光偏振及熒光壽命的影響,來探究AmB/DPPC雙層膜的親水膜區(qū)域和疏水膜區(qū)域受不同金屬離子的影響,結(jié)果表明:(1)比較有和無膽固醇兩種脂質(zhì)體體系,鈉離子環(huán)境下,膽固醇加入后,親水和疏水區(qū)域有序性均降低,鈣離子環(huán)境下恰恰相反,膽固醇加入后,親水和疏水區(qū)域有序性均增大。而鉀離子環(huán)境下,膽固醇的加入,使脂質(zhì)體膜親水區(qū)域有序性增大,疏水區(qū)域有序性減小。對比有和無膽固醇脂質(zhì)體體系中熒光脂的REES值,可知加入膽固醇后,不同條件下均降低,說明膽固醇的加入使脂質(zhì)體中疏水區(qū)�；湹膫�(cè)向包裹能力增大。(2)從某種角度講,這些金屬離子對膽固醇的作用產(chǎn)生了一定的影響。在AmB/Chol/DPPC脂質(zhì)體膜體系中,膽固醇分子與兩性霉素B分子形成復合物,與膜上其他分子相互作用。膽固醇誘導NBD基團向更深區(qū)域定位,說明膽固醇增強了膜的包裹能力。這一現(xiàn)象同時受到鉀、鈉和鈣離子的影響。對于膽固醇對膜親水區(qū)域的影響,鉀離子無明顯作用,鈉離子略有抑制作用,而鈣離子起促進作用。然而,對于膽固醇對疏水區(qū)域的影響,鉀離子起到抑制作用,而鈉離子和鈣離子均起到促進作用。已有報道說明高鈉鹽的攝入可能改善AmB對病人的細胞毒性,這也許與鈉離子對膽固醇增強膜包裹能力的促進作用相關(guān)。(3)這些金屬離子對膽固醇作用的影響也許與其在膜上的離子運輸方式相關(guān)。膽固醇與AmB形成的孔洞具有高鉀離子通透性,膽固醇存在時,鉀離子主動通過膜進入脂質(zhì)體內(nèi)部。但是,鈉離子和鈣離子通過被動運輸由高濃度向低濃度穿過膜進入脂質(zhì)體內(nèi)部。并且,鈣離子具有維持生物膜穩(wěn)定性的特殊作用。兩性霉素B藥物進入人體后,與細胞膜上膽固醇結(jié)合形成孔洞,同時受到細胞內(nèi)不同生命金屬離子的不同影響,這可能與其離子運輸方式不同相關(guān)。
[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.
【學位授予單位】：陜西師范大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R96

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