脂筏微区分子的分离与重组研究
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摘要
目的:提取纯化单唾液酸四己糖神经节苷脂GM_1,研究物理化学因素对四元体系单层膜相变的影响,为脂筏微区分子的分离与重组研究提供理论和实验依据。
方法:1.采用Folch萃取法、DEAE-SephadexA-25离子交换柱层析和硅胶吸附柱层析提取纯化GM_1,通过紫外光谱、红外光谱以及原子力显微镜对其结构进行研究。2.利用LB技术得出不同条件下四元体系单层膜等温线,采用Y型提膜法制备双层膜。3.使用原子力显微镜观测LB双层膜的结构。
结果:1.从1000 g牛脑湿组织中获得GM_139.383 mg,纯度达71.76%,其紫外光谱在195nm处有一吸收峰。通过红外光谱研究及唾液酸含量测定,证明在提纯的产品结构中含有唾液酸分子的结构片段。利用原子力显微镜对其在水中的微观形貌进行了观察研究,在纳米尺度看到了GM_1的疏水部分呈现链状结构,其亲水部分呈现环状结构。
2.改变溶质浓度、亚相温度、GM_1及胆固醇的浓度,得到了不同实验条件下四元体系单层膜等温线,分析了这些因素对脂筏微区超分子聚集体单层膜相变的影响:①溶质的浓度增大,相变提前;②亚相温度升高,相变延迟;③GM_1浓度增加,相变明显提前;④胆固醇的含量增加,第二次相变前后膜压呈现两种相反的变化趋势,前者逐渐增加,后者逐渐减小。
3.对浓度、温度、GM_1含量、胆固醇含量和膜压与相变之间的关系的研究。
①根据相变与浓度和膜压之间的关系,制备了LB双层膜,AFM结果表明,膜压较低时均出现了相分离现象,随着膜压的增加,这种现象完全消失;不同浓度下都出现了细丝网状结构,随着膜压的增加,细丝网状结构排列更加紧密。同种浓度下,随着膜压的增加,组成网状结构细丝的长度和宽度都减小,而随着浓度的增加,相同膜压下细丝的宽度减小,而其长度变化不明显。不同浓度不同膜压下,均出现了微区结构。
②根据相变与温度和膜压之间的关系,制备了LB双层膜,AFM结果表明,膜压较低时均出现了相分离现象,随着膜压的增加,这种现象基本消失。不同温度双层膜中均有细丝结构或由细丝组成的网状结构,且均有微区结构出现,随着温度的升高,微区结构减少。温度为24.5、36.4、45.2℃时,随着膜压的增加,细丝网状结构排列更加紧密;但温度降低到15.3℃时,随着膜压的增加,细丝结构逐渐减少且演化为片层结构。
③根据相变与GM_1含量和膜压之间的关系,制备了LB双层膜,AFM结果表明,当GM_1浓度较小时(1%),较易形成圆形微区结构,膜压为10 mN/m时,有少量较短的条形分枝状结构存在于双层膜中,随着膜压的增z加,条形分枝状结构逐渐消失,微区结构逐渐增加,有些微区间发生了相互融合;当GM_1浓度为5%,双层膜中可以观察到细丝分枝状结构及少量的微区结构,但微区结构有向条形枝状结构转化的趋势,随着膜压的增加,这种趋势更加明显;GM_1浓度为10%,出现了大量的细丝网状结构及少量的微区结构,随着膜压的增加,细丝网状结构排列更加紧密,微区结构出现了相互融合现象。
④根据相变与胆固醇含量和膜压之间的关系,制备了LB双层膜,AFM结果表明,胆固醇含量为10%、20%,膜压为10、30、38 mN/m时双层膜均出现了相分离,双层膜中有大量的条形枝状结构、少量的细丝网状结构及微区结构。膜压为38 mN/m时,双层膜的排列较膜压为30 mN/m时更为疏松。胆固醇含量为20%时,结构的排列较含量为10%时更加紧密。当胆固醇含量为30%时,膜压为10、30 mN/m时均出现了相分离,且有微区结构出现,这时双层膜中出现了大量的细丝网状结构,随着膜压的增加,形成了更加致密的双层膜结构,且有些微区结构发生了相互融合。
结论:溶质浓度、亚相温度、GM_1及胆固醇的浓度对脂筏微区超分子聚集体的相变行为及结构均有一定的影响。当溶质的浓度增大时,亚相上的分子数随之增多,分子间的距离减小,分子间的超分子作用力增强,分子有序性增加,相变提前;温度增加,分子有序簇集体稳定性被减弱,使分子的布朗运动加剧导致体系混乱度(熵)增加,所以分子之间的超分子作用力减小;GM_1多羟基、多酰胺键的特殊结构可与其它组分之间发生较强的氢键作用;胆固醇的双重作用赋予了它特殊的功能。
Aim:In order to supply theoretical and experimental basis for the study which is on the isolation and reconstruction of molecules of lipid rafts microdomains,we extracted and purified the ganglioside GM_1,then we investigated the effect of physical and chemical factors on the phase behaviors of monolayer film of the quattuor system.
Methods:1.The ganglioside GM_1 were extracted from bovine brain by the Folch method and purified by silica gel and DEAE-Sephadex A-25 column chromatograph.Its structure was studied by infrared spectrum(IR) spectra、ultraviolet spectrum(UV) spectra and atomic force microscope(AFM).2.The trough area-surface pressure(π-A) isotherms under different conditions were obtained by LB technology,and the bilayer membranes were prepared by the Y-shaped deposition under certain surface pressure.3.The structure of bilayer membranes were observed by atomic force microcopy.
Results:1.39.383 mg of Gls from 1000 g of wet bovine brain have certain purity,71.76%.And their absorption of Uv spectra appear at 195 nm,near to the results reported by other peoples. Compared with IR spectra of sialic acid and the experiment of content determination of it,the results showed that the structures of the products have the units of sialic acid.In order to investigate the structure of GM_1.AFM technique was applied in water,and the results showed hydrophobic components of GM_1 can form chain structure and hydrophilic components Of it can form ring structure which is formed by several monosaccharides.
2:The trough area-surface pressure isotherms under different condition were obtained through changing the concentration,the content of cholesterol or ganglioside GM_1 and the temperature,and the effects of these factors on the phase behaviors of monolayer film of supramolecular aggregates of lipid raft microdomains were investigated by the LB method:
①When the concentration was increased,the self-assemblies of molecules were strengthened.
②The higher temperature will result in the disorder of the self-assemblied aggregates.As a result,the phase transition will be deferred shown in theπ-A curve.
③When the concentration of ganglioside GM_1 wsa increased,the self-assemblies of molecules were also strengthened.
④With the increase of cholesterol concentration,two opposite trends of film pressure appeared at the point of the second phase transition,before up,and after down.
3:According to the relationship between concentration、temperature、content of GM_1、contnet of GM_1 and membrane pressure、phase transition,the LB bilayer membranes were prepared.
①According to the relationship between phase transition and concentration、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that phase separation phenomenon was found at low membrane pressure under different concentration,and with the increase in membrane pressure,the phenomenon disappears completely.The filament network structure appears under different concentration,and with the increase in membrane pressure,it arranged more closely.When the concentration was same,with the increase in membrane pressure,the length and width of the filaments of the network structure were decreased,and with increasing concentrations,the width of the filaments decreases and its length did not change obviously under the same membrane pressure.The microdomain appeared under different concentration and membrane pressure.
②According to the relationship between phase transition and temperature、membrane pressure, the LB bilayer membranes were prepared.AFM results showed that phase separation phenomenon was found at low membrane pressure under different concentration,and with the increase in membrane pressure,the phenomenon disappeared completely.The filament structure、network structure formed by filaments and microdomains appeared in the bilayer membranes,and with the temperature increase,number of the microdomains decreased.When the temperature was 24.5、36.4、45.2℃,with the increase in membrane pressure,The filament network structure arranged more closely;but when the temperature dropped to 15.3℃,with the increase in pressure membrane,the number of filament structure decreased and evolved in lamellar structure.
③According to the relationship between phase transition and content of GM_1、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that when concentration of GM_1 was smaller(1%),it was easy to form circular microdomains,when the membrane pressure was 10 mN/m,a little shorter strip branching structure existed in the bilayer membrane,and with the membrane pressure increased,the strip branching structure gradually disappeared,microdomains structure gradually increased,some microdomains integrated with each other;when the concentration of GM_1 was 5%,there were some filament branching structures and a small number of microdomains in the bilayer membrane,but the microdomain structure had transformation trend to strip branching structure,as the increase in pressure membrane,this tendency has become even more obvious;when the concentration of GM_1 was 10%,there have been substantial and a small number of microdomain structure,with the increase in membrane pressure,filament network structure arranged more closely,and there has been the phenomenon of the integration of.microdomains.
④According to the relationship between phase transition and content of cholesterol、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that when the content of cholesterol was 10%、20%,membrane pressure was 10、30、38 mN/m,phase separation phenomenon was found,there have been a great number of strip branching structure、a little filament network structure and microdomain.When the membrane pressure was 38 mN/m,the bilayer membrane arranged more loose than 30 mN/m.When the content of cholesterol was 20%,the arrangement of the structure was more closely than the content of 10%.When the content of cholesterol was 30%,membrane pressure was 10、30 mN/m,there has been phase separation phenomenon,microdomains structure was found,and there has been a great number of filament network structure in the bilayer membrane.With increasing the membrane pressure, the bilayer membrane structure was more compact,and there has been the phenomenon of the integration of.microdomains.
Conclusion:The concentration、the temperature of sub-phase、the content of cholesterol or ganglioside GM_1 all have a certain effect on the phase behaviors and structure of supramolecular aggregates of lipid raft microdomains,when the concentration is increased,the molecular number in the sub-phase is increased;the self-assemblies of molecules are strengthened,due to the stronger intermolecular interactions;the higher temperature will result in the disorder of the self-assemblied aggregates,as a result,supramolecular forces between molecules decrease;GM_1 has stronger hydrogen bonding with other molecules in the system;the double role of cholesterol gives its special features.
方法:1.采用Folch萃取法、DEAE-SephadexA-25离子交换柱层析和硅胶吸附柱层析提取纯化GM_1,通过紫外光谱、红外光谱以及原子力显微镜对其结构进行研究。2.利用LB技术得出不同条件下四元体系单层膜等温线,采用Y型提膜法制备双层膜。3.使用原子力显微镜观测LB双层膜的结构。
结果:1.从1000 g牛脑湿组织中获得GM_139.383 mg,纯度达71.76%,其紫外光谱在195nm处有一吸收峰。通过红外光谱研究及唾液酸含量测定,证明在提纯的产品结构中含有唾液酸分子的结构片段。利用原子力显微镜对其在水中的微观形貌进行了观察研究,在纳米尺度看到了GM_1的疏水部分呈现链状结构,其亲水部分呈现环状结构。
2.改变溶质浓度、亚相温度、GM_1及胆固醇的浓度,得到了不同实验条件下四元体系单层膜等温线,分析了这些因素对脂筏微区超分子聚集体单层膜相变的影响:①溶质的浓度增大,相变提前;②亚相温度升高,相变延迟;③GM_1浓度增加,相变明显提前;④胆固醇的含量增加,第二次相变前后膜压呈现两种相反的变化趋势,前者逐渐增加,后者逐渐减小。
3.对浓度、温度、GM_1含量、胆固醇含量和膜压与相变之间的关系的研究。
①根据相变与浓度和膜压之间的关系,制备了LB双层膜,AFM结果表明,膜压较低时均出现了相分离现象,随着膜压的增加,这种现象完全消失;不同浓度下都出现了细丝网状结构,随着膜压的增加,细丝网状结构排列更加紧密。同种浓度下,随着膜压的增加,组成网状结构细丝的长度和宽度都减小,而随着浓度的增加,相同膜压下细丝的宽度减小,而其长度变化不明显。不同浓度不同膜压下,均出现了微区结构。
②根据相变与温度和膜压之间的关系,制备了LB双层膜,AFM结果表明,膜压较低时均出现了相分离现象,随着膜压的增加,这种现象基本消失。不同温度双层膜中均有细丝结构或由细丝组成的网状结构,且均有微区结构出现,随着温度的升高,微区结构减少。温度为24.5、36.4、45.2℃时,随着膜压的增加,细丝网状结构排列更加紧密;但温度降低到15.3℃时,随着膜压的增加,细丝结构逐渐减少且演化为片层结构。
③根据相变与GM_1含量和膜压之间的关系,制备了LB双层膜,AFM结果表明,当GM_1浓度较小时(1%),较易形成圆形微区结构,膜压为10 mN/m时,有少量较短的条形分枝状结构存在于双层膜中,随着膜压的增z加,条形分枝状结构逐渐消失,微区结构逐渐增加,有些微区间发生了相互融合;当GM_1浓度为5%,双层膜中可以观察到细丝分枝状结构及少量的微区结构,但微区结构有向条形枝状结构转化的趋势,随着膜压的增加,这种趋势更加明显;GM_1浓度为10%,出现了大量的细丝网状结构及少量的微区结构,随着膜压的增加,细丝网状结构排列更加紧密,微区结构出现了相互融合现象。
④根据相变与胆固醇含量和膜压之间的关系,制备了LB双层膜,AFM结果表明,胆固醇含量为10%、20%,膜压为10、30、38 mN/m时双层膜均出现了相分离,双层膜中有大量的条形枝状结构、少量的细丝网状结构及微区结构。膜压为38 mN/m时,双层膜的排列较膜压为30 mN/m时更为疏松。胆固醇含量为20%时,结构的排列较含量为10%时更加紧密。当胆固醇含量为30%时,膜压为10、30 mN/m时均出现了相分离,且有微区结构出现,这时双层膜中出现了大量的细丝网状结构,随着膜压的增加,形成了更加致密的双层膜结构,且有些微区结构发生了相互融合。
结论:溶质浓度、亚相温度、GM_1及胆固醇的浓度对脂筏微区超分子聚集体的相变行为及结构均有一定的影响。当溶质的浓度增大时,亚相上的分子数随之增多,分子间的距离减小,分子间的超分子作用力增强,分子有序性增加,相变提前;温度增加,分子有序簇集体稳定性被减弱,使分子的布朗运动加剧导致体系混乱度(熵)增加,所以分子之间的超分子作用力减小;GM_1多羟基、多酰胺键的特殊结构可与其它组分之间发生较强的氢键作用;胆固醇的双重作用赋予了它特殊的功能。
Aim:In order to supply theoretical and experimental basis for the study which is on the isolation and reconstruction of molecules of lipid rafts microdomains,we extracted and purified the ganglioside GM_1,then we investigated the effect of physical and chemical factors on the phase behaviors of monolayer film of the quattuor system.
Methods:1.The ganglioside GM_1 were extracted from bovine brain by the Folch method and purified by silica gel and DEAE-Sephadex A-25 column chromatograph.Its structure was studied by infrared spectrum(IR) spectra、ultraviolet spectrum(UV) spectra and atomic force microscope(AFM).2.The trough area-surface pressure(π-A) isotherms under different conditions were obtained by LB technology,and the bilayer membranes were prepared by the Y-shaped deposition under certain surface pressure.3.The structure of bilayer membranes were observed by atomic force microcopy.
Results:1.39.383 mg of Gls from 1000 g of wet bovine brain have certain purity,71.76%.And their absorption of Uv spectra appear at 195 nm,near to the results reported by other peoples. Compared with IR spectra of sialic acid and the experiment of content determination of it,the results showed that the structures of the products have the units of sialic acid.In order to investigate the structure of GM_1.AFM technique was applied in water,and the results showed hydrophobic components of GM_1 can form chain structure and hydrophilic components Of it can form ring structure which is formed by several monosaccharides.
2:The trough area-surface pressure isotherms under different condition were obtained through changing the concentration,the content of cholesterol or ganglioside GM_1 and the temperature,and the effects of these factors on the phase behaviors of monolayer film of supramolecular aggregates of lipid raft microdomains were investigated by the LB method:
①When the concentration was increased,the self-assemblies of molecules were strengthened.
②The higher temperature will result in the disorder of the self-assemblied aggregates.As a result,the phase transition will be deferred shown in theπ-A curve.
③When the concentration of ganglioside GM_1 wsa increased,the self-assemblies of molecules were also strengthened.
④With the increase of cholesterol concentration,two opposite trends of film pressure appeared at the point of the second phase transition,before up,and after down.
3:According to the relationship between concentration、temperature、content of GM_1、contnet of GM_1 and membrane pressure、phase transition,the LB bilayer membranes were prepared.
①According to the relationship between phase transition and concentration、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that phase separation phenomenon was found at low membrane pressure under different concentration,and with the increase in membrane pressure,the phenomenon disappears completely.The filament network structure appears under different concentration,and with the increase in membrane pressure,it arranged more closely.When the concentration was same,with the increase in membrane pressure,the length and width of the filaments of the network structure were decreased,and with increasing concentrations,the width of the filaments decreases and its length did not change obviously under the same membrane pressure.The microdomain appeared under different concentration and membrane pressure.
②According to the relationship between phase transition and temperature、membrane pressure, the LB bilayer membranes were prepared.AFM results showed that phase separation phenomenon was found at low membrane pressure under different concentration,and with the increase in membrane pressure,the phenomenon disappeared completely.The filament structure、network structure formed by filaments and microdomains appeared in the bilayer membranes,and with the temperature increase,number of the microdomains decreased.When the temperature was 24.5、36.4、45.2℃,with the increase in membrane pressure,The filament network structure arranged more closely;but when the temperature dropped to 15.3℃,with the increase in pressure membrane,the number of filament structure decreased and evolved in lamellar structure.
③According to the relationship between phase transition and content of GM_1、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that when concentration of GM_1 was smaller(1%),it was easy to form circular microdomains,when the membrane pressure was 10 mN/m,a little shorter strip branching structure existed in the bilayer membrane,and with the membrane pressure increased,the strip branching structure gradually disappeared,microdomains structure gradually increased,some microdomains integrated with each other;when the concentration of GM_1 was 5%,there were some filament branching structures and a small number of microdomains in the bilayer membrane,but the microdomain structure had transformation trend to strip branching structure,as the increase in pressure membrane,this tendency has become even more obvious;when the concentration of GM_1 was 10%,there have been substantial and a small number of microdomain structure,with the increase in membrane pressure,filament network structure arranged more closely,and there has been the phenomenon of the integration of.microdomains.
④According to the relationship between phase transition and content of cholesterol、membrane pressure,the LB bilayer membranes were prepared.AFM results showed that when the content of cholesterol was 10%、20%,membrane pressure was 10、30、38 mN/m,phase separation phenomenon was found,there have been a great number of strip branching structure、a little filament network structure and microdomain.When the membrane pressure was 38 mN/m,the bilayer membrane arranged more loose than 30 mN/m.When the content of cholesterol was 20%,the arrangement of the structure was more closely than the content of 10%.When the content of cholesterol was 30%,membrane pressure was 10、30 mN/m,there has been phase separation phenomenon,microdomains structure was found,and there has been a great number of filament network structure in the bilayer membrane.With increasing the membrane pressure, the bilayer membrane structure was more compact,and there has been the phenomenon of the integration of.microdomains.
Conclusion:The concentration、the temperature of sub-phase、the content of cholesterol or ganglioside GM_1 all have a certain effect on the phase behaviors and structure of supramolecular aggregates of lipid raft microdomains,when the concentration is increased,the molecular number in the sub-phase is increased;the self-assemblies of molecules are strengthened,due to the stronger intermolecular interactions;the higher temperature will result in the disorder of the self-assemblied aggregates,as a result,supramolecular forces between molecules decrease;GM_1 has stronger hydrogen bonding with other molecules in the system;the double role of cholesterol gives its special features.
引文
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