Langmuir-Blodgett膜超分子SM/DOPC/Chol三元脂系的原子力显微镜观察

用原子力显微镜研究了胆固醇(Chol)对鞘磷脂(SM)/1,2-二油酸甘油-3-磷脂酰胆碱(DOPC)二元脂系统结构的影响和神经酰胺对SM/DOPC/Chol三元脂系统结构的影响. 实验发现, 在SM/DOPC二元脂系统中, 胆固醇和带饱和脂肪酸链的磷脂发生相互作用形成微区结构, 随着胆固醇含量的增加, 微区的面积逐渐增大, 形成了稳定的片层结构. 当把神经酰胺加入到等摩尔配比的SM/DOPC/Chol三元脂系统中时, 随着神经酰胺比例的增加, 先形成紧密的聚集态结构, 然后逐渐演变成具有特定微区的网状结构. 研究结果表明, 微区的形成主要是由分子不同的官能团之间的相互作用所决定, 这可能在细胞信号传导等生理活动中起到重要的作用.     

DOPC/DPPC单层膜中分子间的相互作用及形态特征

利用Langmuir-Blodgett(LB)技术制备了不同表面压力下的1,2-二油酸-甘油-3-磷脂酰胆碱(DOPC)/1,2-二棕榈酸甘油-3-磷脂酰胆碱(DPPC)(摩尔比为1:1)和DOPC/DPPC/Chol(摩尔比为2:2:1)单层膜, 对单层膜内分子间的相互作用进行了热力学分析, 并用荧光显微镜和原子力显微镜对其形态进行了观测.热力学分析表明, DOPC与DPPC分子在单层膜结构中相互作用为排斥力, 诱导单层膜出现相变; DOPC, DPPC与胆固醇(Chol)间的相互作用均为吸引力, 当表面压力(π)大于18 mN/m时, DPPC与胆固醇的作用力大于DOPC.荧光显微镜观测表明, DOPC/DPPC单层膜出现明显相分离现象, 富含DPPC微区成“花形”结构, 且随着表面压力的升高微区逐渐增大, “花瓣”增多; 当胆固醇加入到DOPC/DPPC体系时, 单层膜相态由液相与凝胶相共存转变为液态无序相与液态有序相共存结构, 富含DPPC的微区形状从“花形”转变成“圆形”.原子力显微镜对单层膜的表征验证了荧光显微镜的观测结果, 表明胆固醇加入到DOPC/DPPC体系中对单层膜排列具有明显的影响, 压力和溶液状态等是影响脂膜结构的重要因素.     

脂筏微区超分子聚集体结构的稳定性

生物膜中脂筏微区结构的动态特征与稳定性决定着生物膜的功能。通过从动物细胞提取脂筏,实验不但观测到质膜微囊烧瓶状凹陷结构,而且还观测到大量的球状和椭球状结构.通过模拟脂筏微区结构,重点对二元体系和三元体系的超分子聚集体结构的多形性进行了研究和探索。研究发现随着表面压力的增加,鞘磷脂和胆固醇双层膜出现了紧密聚集不规则的微区结构,在 SM/Chol/DOPC双层膜中,SM/Chol形成的微区结构漂浮在液态DOPC小颗粒上部。当 DOPE加入到SM/Chol中,三种成份形成不稳定的双层膜结构.Ceramide促进了SM/Chol结构发生重排,微区形状从原来的不规则向着紧密聚集的圆形结构演变;混合单层膜的分子面积与表面吉布斯自由能决定了分子间的相互作用, 当过量分子面积与过量吉布斯自由能为负值时,分子间相互作用表现为吸引力, 出现凝聚现象; 为正值时,分子间相互作用表现为排斥力, 促使单层膜出现相分离现象. 过量吉布斯自由能值越小, 单层膜的热稳定性越高.通过动物细胞提取脂筏与体外模拟脂筏相结合的方法,从超分子水平阐述了脂筏微区结构与功能的生物学意义,为生物膜的研究提供了理论依据和实验支持。     

衍生于咔唑的双氰基二苯代乙烯型双光子荧光脂筏探针

制备了一个衍生于咔唑的双氰基二苯代乙烯型双光子荧光脂筏探针——(E)-2-甲基-5-{2-[9-正辛基(3-咔唑基)]乙烯基}对苯二甲腈(DLR), 并对其结构进行了表征. 结果表明, DLR属于推-拉电子结构(供体-桥-受体, D-π-A), 其最大发射波长随介质极性递增, 而其荧光强度却随极性递减. DLR在二棕榈酰磷脂酰胆碱 (DPPC)中的发射强度是在二油酰磷脂酰胆碱(DOPC)中的20倍, 其对DPPC, 模拟脂筏[n(DOPC)∶n(鞘磷脂)∶n(胆固醇)=1∶1∶1]和DOPC的荧光强度比为20∶12.8∶1, 在DPPC中的荧光寿命是在DOPC中的2.2倍以上, 表明DLR能很好地区分DPPC与DOPC. DLR在DPPC和DOPC中的双光子发射截面(Φδ)分别为1350和67 GM, 表明DLR能够很好地识别脂筏, 成像脂筏在细胞与组织中的分布动态.     

糖和盐类物质对生物膜超分子结构稳定性影响的研究

用原子力显微镜(AFM)和小角X射线(SAXS)技术, 研究了NaCl、KCl、胆固醇、葡萄糖和蔗糖等与膜脂的相互作用. 研究发现它们能引起脂质膜超分子体系液晶态结构的变化. 葡萄糖和蔗糖对脂双层膜结构有稳定作用. 在NaCl溶液中制成的脂质膜, 随着NaCl浓度的增加, 它们的双层膜更稳定. 在KCl溶液中结果恰好相反. AFM研究发现液晶态脂双层膜结构与双亲性分子的结构、浓度以及介质的组分和pH等因素有关. 在1,2-反十八碳-3-磷脂酰乙醇胺(DEPE)液晶态中, 钠盐诱导形成Q²²⁹(Im3m)立方相. 油酸的含量对DEPE-PVP(聚乙烯吡咯烷酮)超分子结构也有一定的影响, 当油酸含量达到某一临界值时, 则发生从Im3m(Q²²⁹)到Pn3m(Q²²⁴)的转变. 胆固醇能促使形成Pn3m(Q²²⁴)和六角相H_II共存相. 研究结果表明, 生物膜超分子聚集体的氢键、分子van der Waals力、离子的静电力等这些弱相互作用的协同性、方向性和选择性, 可能决定着生物膜的结构和功能.     

不同固醇与DPPC二元体系的液态有序相

应用同步辐射X射线衍射和差示扫描量热法研究了由不同结构的固醇(胆固醇、脱氢胆固醇、豆固醇、谷固醇、麦角固醇以及固醇核)和二棕榈酰磷脂酰胆碱(DPPC)二元体系形成的液态有序相. 研究表明, 胆固醇比植物固醇(豆固醇和谷固醇)和真菌固醇(麦角固醇)能更有效地与DPPC形成液态有序相(Lo); 有胆固醇或者脱氢胆固醇参与的液态有序相能够在较宽的温度范围内保持稳定, 而由植物固醇和真菌固醇参与的液态有序相对温度有较强的依赖性, 在DPPC主相变温度附近有明显的热致相变过程, 因此这一液态有序相应该进一步区分为Loβ和Loα相. 研究结果有助于阐明固醇尾链在液态有序相以及脂筏中的作用, 也有助于理解在进化过程中动物细胞膜为何选择胆固醇作为主要固醇.     

巨型囊泡的侧向相分离与出芽

用电形成法制备含三组分二油酰磷脂酰胆碱(DOPC)/二棕榈酰磷脂酰胆碱(DPPC)/胆固醇(Chol)的巨型囊泡, 以TR-DPPE为荧光染色剂, 在荧光显微镜下直接观察膜的侧向相分离与微区相凸起出芽的耦合. 发现囊泡膜内的相分离具有诱导期, 相分离速度很快, 形成的微相区在整个囊泡球面上均衡分布. 各微相区的出芽不是同时进行, 为逐个随机发生. 每次出芽的时间小于0.5 s. 分相与出芽的耦合使球面上的不同微区之间不会相互融合成更大的微区.     

二棕榈酰磷脂酰胆碱/豆固醇脂质体液态有序相的结构性质:同步辐射X光衍射研究

用同步辐射小角和宽角X光衍射实验技术研究了由二棕榈酰磷脂酰胆碱(DPPC)和豆固醇所形成的脂质体的液态有序相的结构性质. 结果表明液态有序相的小角X光衍射d值(d-spacing)随着固醇温度和浓度的变化仅有微小的改变. 与凝胶相及液晶相的宽角X光衍射d值相比, 液态有序相的宽角X光衍射d值有更宽的变化范围, 在30到52 °C的温度范围内, 液态有序相的宽角X光衍射d值从0.422 nm变化到0.460 nm. 电子云密度计算表明液态有序相的脂双层厚度和水层厚度都要大于与之平衡共存的液晶相的脂双层厚度和水层厚度. 电子云密度计算结果还表明液态有序相的脂双层厚度随温度升高而降低. 本研究结果对于从定量的角度认识 生物膜的相态及深入认识生物膜中的有序结构具有重要意义.     

脂双层膜表面结构与稳定性的原子力显微镜研究

用原子力显微镜研究了1,2－二油酸甘油－3－磷酸－1甘油（DOPG）脂双层膜 的表面结构与稳定性。实验结果表明,原子力显微镜的探针与脂双层膜的相互作用 导致脂双层膜表面产生一个永久的损伤。静电相互作用对脂双层膜结构和稳定性的 影响表明,在NaCl溶液中制成的脂质体,随着NaCl浓度的增加,它们的双层膜更稳 定。在低的NaCl浓度则经常被损伤,在1 mol/L NaCl溶液中制备的指双层变得更稳 定。在KCl溶液中结果恰好相反。在高的KCl浓度中经常被损伤,随着KCl浓度的降 低,它们的双层膜更稳定。葡萄糖和蔗糖对脂双层膜结构有稳定作用。     

脂质体药用辅料DMPG的合成研究

以三氯氧磷、1,2-二肉豆蔻酰甘油及甘油缩丙酮为原料,通过磷酰化、水解等反应合成1,2-二肉豆蔻酰磷脂酰甘油(DMPG),反应收率为75.8%,其结构经过IR,MS,~1H NMR及~(13)C NMR等确证。该合成方法具有操作简单、条件温和、收率高等特点。     

Structure of a gel phase lipid bilayer prepared by the Langmuir-Blodgett/Langmuir-Schaefer method characterized by sum-frequency vibrational spectroscopy

The structure of a planar supported lipid bilayer (PSLB) prepared by the Langmuir-Blodgett (LB)/Langmuir-Schaefer (LS) method was investigated by sum-frequency vibrational spectroscopy (SFVS). By using asymmetric lipid bilayers composed of selectively deuterated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) lipids, the orientation of the fatty acid chains and phosphocholine headgroups has been determined independently for both leaflets of the bilayer. The alkyl chains of the lipids were found to be orientated approximately 13 degrees +/- 4 degrees from the surface normal for both leaflets. The lipid chains in both leaflets also contain some gauche content, which is consistent with previous NMR and FTIR studies of similar lipid systems. More importantly, the relative number of gauche defects does not seem to be influenced by the deposition method, LB versus LS. The headgroup orientation for the lipid film in contact with the silica support was determined to be 69 degrees +/- 3 degrees , whereas that in contact with the aqueous phase was 66 degrees +/- 4 degrees from the surface normal. The SFVS results indicate that the structure of the DSPC lipid film in contact with the solid support and the film adjacent to the aqueous phase are nearly identical in structure. These results suggesting the LB/LS deposition method do indeed produce symmetric lipid bilayers. These studies further add to the growing information on the efficacy of PSLBs as suitable models for biological membrane studies.     

Measuring lipid asymmetry in planar supported bilayers by fluorescence interference contrast microscopy

There is substantial scientific and practical interest in engineering supported lipid bilayers with asymmetric lipid distributions as models for biological cell membranes. In principle, it should be possible to make asymmetric supported lipid bilayers by either the Langmuir-Blodgett/Schafer (LB/LS) or Langmuir-Blodgett/vesicle fusion (LB/VF) techniques (Kalb et al. Biochim. Biophys. Acta 1992, 1103, 307-316). However, the retention of asymmetry in biologically relevant lipid bilayers has never been experimentally examined in any of these systems. In the present work, we developed a technique that is based on fluorescence interference contrast (FLIC) microscopy to measure lipid asymmetry in supported bilayers. We compared the final degree of lipid asymmetry in LB/LS and LB/VF bilayers with and without cholesterol in liquid-ordered (l(o)) and liquid-disordered (l(d)) phases. Of five different fluorescent lipid probes that were examined, 1,2-dipalmitoyl-phosphatidylethanolamine-N-[lissamine rhodamine B] was the best for studying supported bilayers of complex composition and phase by FLIC microscopy. An asymmetrically labeled bilayer made by the LB/LS method was found to be at best 70-80% asymmetric once completed. In LB/LS bilayers of either l(o) or l(d) phase, cholesterol increased the degree of lipid mixing between the opposing monolayers. The use of a tethered polymer support for the initial monolayer did not improve lipid asymmetry in the resulting bilayer. However, asymmetric LB/VF bilayers retained nearly 100% asymmetric label, with or without the use of a tethered polymer support. Finally, lipid mixing across the center of LB/LS bilayers was found to have drastic effects on the appearance of l(d)-l(o) phase coexistence as shown by epifluorescence microscopy.     

Photopolymerization of mixed monolayers and black lipid membranes containing gramicidin A and diacetylenic phospholipids

We formed monolayers and black lipid membranes (BLMs) of photopolymerizable lipids mixed with the channel-forming protein gramicidin A to evaluate their miscibility and the potential for improved stability of the BLM scaffold through polymerization. Analyses of surface pressure vs area isotherms indicated that gramicidin A dispersed with three different synthetic, polymerizable, diacetylene-containing phospholipids, 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-di-10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (DTPE), and 1-palmitoyl-2,10,12-tricosadiynoyl-sn-glycero-3-phosphoethanolamine (PTPE) to form mixed monolayers at the air-water interface on a Langmuir-Blodgett (LB) trough. Conductance measurements across a diacetylenic lipid-containing BLM confirmed dispersion of the gramicidin channel with the lipid layer and demonstrated gramicidin ion-channel activity before and after UV exposure. Polymerization kinetics of the diacetylenic films were monitored by film pressure changes at constant LB trough area and by UV-vis absorption spectroscopy of polymerized monolayers deposited onto quartz. An initial increase in film pressure of both the pure diacetylene lipid monolayers and mixed films upon exposure to UV light indicated a change in the film structure. Over the time scale of the pressure increase, an absorbance peak indicative of polymerization evolved, suggesting that the structural change in the lipid monolayer was due to polymerization. Film pressure and absorbance kinetics also revealed degradation of the polymerized chains at long exposure times, indicating an optimum time of UV irradiation for maximized polymerization in the lipid layer. Accordingly, exposure of polymerizable lipid-containing black lipid membranes to short increments of UV light led to an increase in the bilayer lifetime.     

Dissimilar interaction of CB1/CB2 with lipid bilayers as revealed by molecular dynamics simulation

Cannabinoid receptors CB1 and CB2 are a striking class of transmembrane proteins involved in a high number of important biological processes. In spite of the inherent similarity (40% in aminoacid sequence) these receptors are found in different cell environments. In addition to this, CB1 activity has been intimately associated with lipid rafts whereas CB2 has not. In this work we have performed a 50 nanosecond molecular dynamics simulation of the inactive conformations of both receptors inserted in a POPC lipid bilayer. Although in both cases the overall protein structure is maintained along the entire simulation we have found important differences in the protein-lipid interaction. While CB1 tends to distort the lipid bilayer regularity, especially in the extracellular moiety, CB2 has a minor influence on the lipid distribution along the plane of the bilayer. This observation is consistent with some experimental facts observed in these cannabinoid receptors with regard to lipid/protein interaction.     

聚乙烯醇缩丁醛LB膜制备及其对硬脂酸镉LB膜的修饰

研究了PVB单分子膜的π～A曲线和稳定性,沉积了优质LB膜,发现将PVB与CdSt₂形成交替异质的LB膜能提高膜的光学质量;在CdSt₂LB膜的上面封装PVBLB膜能保持CdSt₂LB膜的结构。     

AFM studies of solid-supported lipid bilayers formed at a Au(111) electrode surface using vesicle fusion and a combination of Langmuir-Blodgett and Langmuir-Schaefer techniques

Atomic force microscopy (AFM) has been used to characterize the formation of a phospholipid bilayer composed of 1,2-dimyristyl-sn-glycero-3-phosphocholine (DMPC) at a Au(111) electrode surface. The bilayer was formed by one of two methods: fusion of lamellar vesicles or by the combination of Langmuir-Blodgett (LB) and Langmuir-Schaefer (LS) deposition. Results indicate that phospholipid vesicles rapidly adsorb and fuse to form a film at the electrode surface. The resulting film undergoes a very slow structural transformation until a characteristic corrugated phase is formed. Force-distance curve measurements reveal that the thickness of the corrugated phase is consistent with the thickness of a bilayer lipid membrane. The formation of the corrugated phase may be explained by considering the elastic properties of the film and taking into account spontaneous curvature induced by the asymmetric environment of the bilayer, in which one side faces the gold substrate and the other side faces the solution. The effect of temperature and electrode potential on the stability of the corrugated phase has also been described.