电制备巨囊泡的方法和机理探索

利用倒置显微镜研究电制备巨囊泡,并分析其形成和成长的动力学机理.由于磷脂头部的电特性,电场对水化的磷脂双层的静电力促使磷脂膜内双层之间分离.在动力学的作用下,双层两叶的不对称受力引发其弯曲、出芽、膨胀、封闭以及相互融合.结果表明,电场参数、干磷脂膜的均匀性、缓冲液以及温度等因素影响巨囊泡的粒径、形状和稳定性.     

蜂毒肽浓度和磷脂组分对巨囊泡泄露的影响

蜂毒肽作为一种广谱抗菌肽已经得到广泛认知,用蜂毒肽构建载药体系攻击癌细胞研究正在兴起.基于仿生物膜模型探索其破坏机理,可以避免潜在活性细胞过程的影响.在此,我们选用细胞尺寸的单层巨囊泡膜模型,可在光学显微镜下直接观察和操作,获得仿正常细胞膜和仿癌细胞膜在不同蜂毒肽浓度刺激下的响应.研究得出,低浓度蜂毒肽诱导囊泡泄露实验表明中性磷脂囊泡以孔模式为主泄露,负电性磷脂囊泡以爆裂模式为主泄露;高浓度蜂毒肽诱导囊泡泄露实验表明负电性磷脂相较于中性磷脂可延迟蜂毒肽作用效果;蜂毒肽色氨酸残基荧光光谱表明囊泡膜表面蜂毒肽吸附量以及泄露模式依赖于磷脂组分.此外,推断了蜂毒肽对不同组分磷脂膜的破坏作用模型.研究为蜂毒肽在肿瘤细胞的作用机制及其衍生物的优化设计提供参考.     

蜂毒肽浓度和磷脂组分对巨囊泡泄露的影响

蜂毒肽作为一种广谱抗菌肽已经得到广泛认知,用蜂毒肽构建载药体系攻击癌细胞研究正在兴起.基于仿生物膜模型探索其破坏机理,可以避免潜在活性细胞过程的影响.在此,我们选用细胞尺寸的单层巨囊泡膜模型,可在光学显微镜下直接观察和操作,获得仿正常细胞膜和仿癌细胞膜在不同蜂毒肽浓度刺激下的响应.研究得出,低浓度蜂毒肽诱导囊泡泄露实验表明中性磷脂囊泡以孔模式为主泄露,负电性磷脂囊泡以爆裂模式为主泄露;高浓度蜂毒肽诱导囊泡泄露实验表明负电性磷脂相较于中性磷脂可延迟蜂毒肽作用效果;蜂毒肽色氨酸残基荧光光谱表明囊泡膜表面蜂毒肽吸附量以及泄露模式依赖于磷脂组分.此外,推断了蜂毒肽对不同组分磷脂膜的破坏作用模型.研究为蜂毒肽在肿瘤细胞的作用机制及其衍生物的优化设计提供参考.     

蜂毒肽与多组分生物膜作用：电性与相态的影响

蜂毒肽非特异性地靶向杀伤细菌具有重要的生物医学应用前景. 利用荧光光谱与荧光显微考察了蜂毒肽与单组分、多组分磷脂膜的作用机制. 发现对于不同电性与相态的磷脂膜, 肽-膜作用呈现为稳定桶板型孔、非稳U型孔及变薄裂解等多种机制, 具有显著不同的内含物泄露效率. 多组分磷脂囊泡的泄露实验表明, 泄露由肽亲和性较强的磷脂组分决定. 相较于凝胶相磷脂, 蜂毒肽与液相磷脂的亲和性强, 凝胶-液相混合囊泡与纯液相磷脂囊泡的泄露性质相近; 相较于双电性磷脂, 蜂毒肽与负电性磷脂的亲和性强, 双电-负电混合囊泡与纯负电磷脂囊泡的泄露性质相近. 研究深化了多肽与多组分生物膜作用机制的理解.     

多聚赖氨酸诱导的负电性磷脂巨囊泡形变

利用荧光显微技术表征了多聚赖氨酸诱导的负电性磷脂巨囊泡的动力学响应行为.研究发现,多聚赖氨酸可吸附至二油酰磷脂酰胆碱和二油酰磷脂酸混合磷脂巨囊泡的表面,诱导其发生粘连、出"绳"及破裂现象.分析认为,在低盐环境中,膜形变由多聚赖氨酸吸附于二油酰磷脂酸富集区引起的膜两叶应力不对称,以及静电相互作用等因素产生.研究结果对基于聚合物-巨囊泡体系的药物输运控释、细胞形变、微控反应和基因治疗等方面的研究提供有价值的支持.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

摘要：巨囊泡作为细胞的简化模型,其分相与出芽机理及动力学规律已引起许多领域科学家的关注。在富含胆固醇的典型生物膜体系如二棕榈酰磷脂酰胆碱DPPC(2-dihexadecanoyl-rac-glycero-3phosphocholine)/二油酰磷脂酰胆碱DOPC(dioleoyl-phosphatidylcholine)/胆固醇(Chol)的三组分形成的巨囊泡作为模型,从高温退火至低温会发生相分离,形成微畴。实验中借助荧光显微镜观察生物膜体系侧向分离的相结构图。实验发现,体系各组分的不同会影响磷脂膜的相结构和膜内微畴的成长,固定 DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇参入量的增加而变大。最后运用理论进一步分析了微畴的成长机理。     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

巨囊泡作为细胞的简化模型,其相分离与出芽动力学规律已引起许多领域科学家的关注.本实验采用DPPC/DOPC/Chol的三组分形成的巨囊泡作为模型,借助荧光显微镜观察该三组分体系侧向分离的相结构图,并对微畴的成长过程作了系统的观察研究和理论分析.实验发现:从高温的均相区域淬灭到低温的分相区域,膜表面发生侧向分离形成微畴.体系内胆固醇的掺入量的多少会影响磷脂膜的相结构和膜内微畴的成长,固定DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇掺入量的增加而变大.     

磷脂跨膜交换机制的研究

膜间磷脂交换是一项重要的生理活动, 其对药物运输及膜功能研究有重要意义. 本文用石英晶体微天平及耗散系数测试仪研究囊泡与囊泡、囊泡与支撑膜间磷脂交换行为, 荧光光谱仪用来测量膜表面电性与膜组分对磷脂交换的影响. 实验结果表明: 磷脂跨膜交换速率与交换时间成反比, 膜表面异电性磷脂的增加会加速膜内相互作用和磷脂跨膜交换速率, 以及改变膜表面组分会对囊泡与支撑膜间的磷脂交换产生影响. 本文研究有助于加深理解磷脂跨膜交换机制, 并对药学研究提供参考.     

磷脂跨膜交换机制的研究

膜间磷脂交换是一项重要的生理活动,其对药物运输及膜功能研究有重要意义.本文用石英晶体微天平及耗散系数测试仪研究囊泡与囊泡、囊泡与支撑膜间磷脂交换行为,荧光光谱仪用来测量膜表面电性与膜组分对磷脂交换的影响.实验结果表明:磷脂跨膜交换速率与交换时间成反比,膜表面异电性磷脂的增加会加速膜内相互作用和磷脂跨膜交换速率,以及改变膜表面组分会对囊泡与支撑膜间的磷脂交换产生影响.本文研究有助于加深理解磷脂跨膜交换机制,并对药学研究提供参考.     

荧光显微镜研究极端pH值诱导磷脂支撑膜的侧向再组织

利用荧光显微镜研究了极端pH值诱导支撑磷脂双层膜的侧向再组织.结果表明,在强酸/强碱性溶液中,流动性较好的二油酰磷脂酰胆碱支撑膜出现破裂、分离、出芽或生出微管等与细胞内吞和外排相似的现象.基于极性分子与H+/H3O+或OH-的相互作用,以电中性的磷脂首基为核吸附溶液中的H+/H3O+或OH-.当磷脂膜上下叶吸附的电荷量不同时,引起两叶有效面积差,即磷脂膜曲率不对称,从而诱发磷脂膜出现各种结构和动力学的响应.本研究有助于理解极端环境对生物膜的影响,为研究生物膜的形变过程提供了参考.     

Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at 94 GHz

In this paper, we report our initial results on studying magnetically aligned phospholipid bilayers (bicelles) at high magnetic fields (approximately 3.4 T) with electron paramagnetic resonance (EPR) spectroscopy at 95 GHz (W-band). In order to characterize this system for W-band EPR studies, we have utilized the nitroxide spin probe 3beta-doxyl-5alpha-cholestane to demonstrate the effects of macroscopic bilayer alignment. At W-band due to the increase in magnetic field strength (when compared to X-band studies at 9.5 GHz) (S. M. Garber et al., J. Am. Chem. Soc. 121, 3240-3241 (1999)), we were able to examine magnetically aligned phospholipid bilayers at two orientations with the bilayer normal oriented either perpendicular or parallel (upon addition of YbCl3) with respect to the direction of the static magnetic field. Additionally, at a magnetic field of 3.4 T (g=2 resonance at W-band), we were able to study the parallel alignment with a lower concentration of Yb3+, thereby eliminating the possible unwanted effects associated with lanthanide-protein interactions and paramagnetic shifts and/or line broadening induced by the lanthanide ions. The development of this new spin label alignment technique will open up a whole new area of investigation for phospholipid bilayer systems and membrane protein EPR studies at high magnetic fields.     

Calculating order parameter profiles utilizing magnetically aligned phospholipid bilayers for 2H solid-state NMR studies

Solid-state deuterium NMR spectroscopy was used to study the structural and dynamic properties of stearic acid-d(35) in magnetically aligned phospholipid bilayers as a function of temperature. Magnetically aligned phospholipid bilayers or bicelles are model systems, which mimic biological membranes for magnetic resonance studies. Paramagnetic lanthanide ions (Yb(3+)) were added to align the bicelles such that the bilayer normal is colinear with the direction of the static magnetic field. The corresponding order parameters of the stearic acid-d(35) probe were calculated and compared with values obtained from unoriented samples in the literature. The addition of cholesterol to the bicelle system decreases the fluidity of the phospholipid bilayers and increases the ordering of the acyl chains of stearic acid-d(35). This study demonstrates the feasibility of utilizing magnetically aligned bicelles for calculating 2H order parameter profiles for non-biological systems such as polymer-grafted membranes and Schiff's base complexes.     

Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy

Solid-state NMR experiments on mechanically aligned bilayer and magnetically aligned bicelle samples demonstrate that membrane proteins undergo rapid rotational diffusion about the normal in phospholipid bilayers. Narrow single-line resonances are observed from 15N labeled sites in the trans-membrane helix of the channel-forming domain of the protein Vpu from HIV-1 in phospholipid bilayers with their normals at angles of 0 degrees, 20 degrees, 40 degrees, and 90 degrees, and bicelles with their normals at angles of 0 degrees and 90 degrees with respect to the direction of the applied magnetic field. This could only occur if the entire polypeptide undergoes rotational diffusion about the bilayer normal. Comparisons between experimental and simulated spectra are consistent with a rotational diffusion coefficient (DR) of approximately 10(5)s-1.     

Fluorescence of Supported Phospholipid Bilayers Recorded in a Conventional Horizontal-Beam Spectrofluorometer

Supported phospholipid bilayers are a convenient model of cellular membranes in studies of membrane biophysics and protein-lipid interactions. Traditionally, supported lipid bilayers are formed on a flat surface of a glass slide to be observed through fluorescence microscopes. This paper describes a method to enable fluorescence detection from the supported lipid bilayers using standard horizontal-beam spectrofluorometers instead of the microscopes. In the proposed approach, the supported lipid bilayers are formed on the inner optical surfaces of the standard fluorescence microcell. To enable observation of the bilayer absorbed on the cell wall, the microcell is placed in a standard fluorometer cell holder and specifically oriented to expose the inner cell walls to both excitation and emission channels with a help of the custom cell adaptor. The signal intensity from supported bilayers doped with 1 % (mol) of rhodamine-labeled lipid in the standard 3-mm optical microcell was equivalent to fluorescence of the 70–80 nM reference solution of rhodamine recorded in a commercial microcell adaptor. Because no modifications to the instruments are required in this method, a variety of steady-state and time-domain fluorescence measurements of the supported phospholipid bilayers may be performed with the spectral resolution using standard horizontal-beam spectrofluorometers.     

Investigating magnetically aligned phospholipid bilayers with EPR spectroscopy at Q-band (35 GHz): optimization and comparison with X-band (9 GHz)

This paper presents the improvement and advantages of investigating magnetically aligned phospholipid bilayers (bicelles) utilizing electron paramagnetic resonance (EPR) spectroscopy at a microwave frequency of 35 GHz (Q-band) and at a high magnetic field strength of 1.25 T when compared to weaker magnetic fields for X-band EPR studies. The nitroxide spin label 3beta-doxyl-5alpha-cholestane (cholestane or CLS) was inserted into the bicelles and utilized to demonstrate the effects of macroscopic bilayer alignment through the measurement of orientational dependent hyperfine splittings. The effects of different lanthanide ions with varying degree of magnetic susceptibility anisotropy were examined. The requirement of minimal amounts of the Tm3+ and Dy3+ lanthanide ions for well-aligned bicelles were examined for Q-band and compared with amounts required for X-band bicelle alignment studies. At a magnetic field of 1.25 T (when compared to 0.63 T at X-band), the perpendicular and parallel orientation were aligned with lower concentrations of Dy3+ and Tm3+, respectively, and thereby eliminating/minimizing the unwanted effects associated with lanthanide-protein interactions. Thus, it is much easier to magnetically align phospholipid bilayers at Q-band when compared to X-band.     

The effects of cholesterol on magnetically aligned phospholipid bilayers: a solid-state NMR and EPR spectroscopy study

This paper presents the first time that both solid-state NMR spectroscopy and EPR spectroscopy are used to study the effects of cholesterol on magnetically aligned phospholipid bilayers (bicelles). Solid-state deuterium NMR spectroscopy was carried out using both chain perdeuterated 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d(54)) and a partially deuterated beta-[2,2,3,4,4,6-(2)H(6)]cholesterol (cholesterol-d(6)). Also, EPR spectroscopy was carried out utilizing a 3 beta-doxyl-5 alpha-cholestane (cholestane) spin probe incorporated into magnetically aligned bilayers to provide a more complete picture about the ordering and dynamics of the phospholipid and cholesterol molecules in the bicelle membrane system. The results demonstrate that cholesterol was successfully incorporated into the phospholipid bilayers. The molecular order parameters extracted directly from the (2)H NMR spectra of both DMPC-d(54) and cholesterol-d(6) were compared to that from the EPR study of cholestane. The order parameters indicate that the sterol was motionally restricted, and that the DMPC had high order and low motion for the hydrocarbon segments close to the head groups of the phospholipids and less order and more rapid motion toward the terminal methyl groups. Both methods clearly indicate an overall increase in the degree of ordering of the molecules in the presence of cholesterol and a decrease in the degree of ordering at higher temperatures. However, EPR spectroscopy and (2)H NMR spectroscopy exhibit different degrees of sensitivity in detecting the phospholipid molecular motions in the membrane. Finally, cholesterol increases the minimum alignment temperature necessary to magnetically align the phospholipid bilayers.     

Solid-state NMR spectroscopy of a membrane protein in biphenyl phospholipid bicelles with the bilayer normal parallel to the magnetic field

Bicelles composed of the long-chain biphenyl phospholipid TBBPC (1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-sn-glycero-3-PC) and the short-chain phospholipid DHPC align with their bilayer normals parallel to the direction of the magnetic field. In contrast, in typical bicelles the long-chain phospholipid is DMPC or DPPC, and the bilayers align with their normals perpendicular to the field. Samples of the membrane-bound form of the major coat protein of Pf1 bacteriophage in TBBPC bicelles are stable for several months, align magnetically over a wide range of temperatures, and yield well-resolved solid-state NMR spectra similar to those obtained from samples aligned mechanically on glass plates or in DMPC bicelle samples "flipped" with lanthanide ions so that their bilayer normals are parallel to the field. The order parameter of the TBBPC bicelle sample decreases from approximately 0.9 to 0.8 upon increasing the temperature from 20 degrees C to 60 degrees C. Since the frequency spans of the chemical shift and dipolar coupling interactions are twice as large as those obtained from proteins in DMPC bicelles without lanthanide ions, TBBPC bicelles provide an opportunity for structural studies with higher spectral resolution of the metal-binding membrane proteins without the risk of chemical or spectroscopic interference from the added lanthanide ions. In addition, the large temperature range of these samples is advantageous for the studies of membrane proteins that are unstable at elevated temperatures and for experiments requiring measurements as a function of temperature.     

tBid蛋白引发磷脂膜透化过程的研究

Bid蛋白是仅有BH3结构域的Bcl-2家族蛋白,在溶酶体膜透化以及线粒体外膜透化引发的细胞凋亡过程中起着非常重要的调控作用,但是Bid蛋白与生物膜之间的相互作用导致脂膜透化的确切机制尚不十分清楚.本文利用激光扫描共聚焦显微成像技术及基于氧化石墨烯表面诱导荧光衰逝的单分子荧光技术,分别从单囊泡及单分子水平对tBid蛋白与磷脂膜之间的相互作用进行了系统的研究.结果表明,tBid蛋白在膜上聚集后可引起脂膜的透化,且脂膜透化的发生源于聚集体中一些tBid蛋白更深入地插入了脂膜中.