猪肺胞膜窖结构的分离与鉴定及与人工膜相互作用的初步研究

发布时间：2018-03-15 08:16

本文选题：胞膜窖　切入点：去污剂法　出处：《陕西师范大学》2011年硕士论文　论文类型：学位论文

【摘要】：胞膜窖(Caveolae)是存在于细胞膜表面大小约为50-100nm的多样化凹陷结构,信号转导过程所涉及的信号蛋白大多都富集于上,行使多种生物学功能,是细胞的信号处理中心,主要承载抑制信号转导作用。窖蛋白(Caveolin)为胞膜窖上特异性标志蛋白,当细胞处于稳态或是由配体引导的信号行为时,可通过形成脚手架区域(scaffold)固定各种信号分子。作为质膜上的永久的生物膜结构,胞膜窖及窖蛋白生理功能的研究逐渐受到人们的重视,尤其在信号转导、胆固醇的运输、细胞内化及肿瘤抑制、肌细胞合成等方面。胞膜窖结构在细胞生命活动过程中起着重要作用,具有重要的的研究价值。本文对胞膜窖结构从两方面进行了研究：(1)胞膜窖结构的分离纯化及生物学鉴定；(2)胞膜窖结构与体外人工模拟单层膜相互作用的初步研究。通过着手这两方面的研究工作,可以为胞膜窖结构及功能的进一步研究打下基础,具有重要意义。实验结果如下： (1)胞膜窖结构的分离纯化及生物学鉴定：实验利用4℃条件下Caveolae不溶于非离子去垢剂ThritonX-100以及所在膜区域具有独特的浮力密度特性的原理,从猪肺中分离得到Caveolae。通过SDS-PAGE可得到特征蛋白条带,利用特异性标志蛋白Caveolin-1抗体进行点杂交实验检测信号蛋白,结果表明Caveolae中含有Caveolin蛋白。考马斯亮蓝法得到样品蛋白含量为0.79113mg/mL。再对纯化出的Caveolae作不同浓度的稀释,通过原子力显微镜得到了清晰的Caveolae结构图,图片结果显示Caveolae的大小为79 nm。最终实验结果表明用该种方法得到的Caveolae和Caveolin蛋白重显性好、结果稳定,可以应用于各种生物组织及细胞系,建立了一种高效快速分离完整Caveolae并用点杂交鉴定Caveolae的新方法。 (2)胞膜窖结构与体外人工模拟单层膜相互作用的初步研究：为了研究细胞膜上组成胞膜窖结构各种脂质组分之间的相互作用机理,我们进行了体外模拟试验。通过LB膜仪,利用表面压力-平均分子面积(π-A)曲线的测量和原子力显微镜观测,研究了二棕榈酰基磷脂酰胆碱(DPPC)和胆固醇(Cholesterol)二元混合体系单层膜在空气/水界面上的热力学特性及AFM形态学特征。π-A曲线分析结果表明,当温度控制在20(±0.5)℃,DPPC的摩尔分数(XDPPC)不变时,随着表面压力(π)的增加,二元混合系统的面积(A)不断减小；当表面压力(π)不变时,随着DPPC的摩尔分数(XDPPC)的增大,在膜压为15mN/m时,二元混合系统的面积(A)出现了先减小后增大的趋势；但在膜压为20mN/m、25mN/m、30mN/m时,二元混合系统的面积(A)出现了非线性的周期性减小；通过吉布斯能量分析,当XDPPC=0.7处,混合体系均出现了负偏差的极大值,当膜压在25mN/m、30mN/m时,DPPC/Chol二元体系在XDPPC=0.1出现正偏差趋势。理论上给出了该体系相互作用随π和XDPPC变化的特征。AFM的观测也给出了直观的证据。最后,我们选取不同比例条件下将纯化出来的胞膜窖结构与模拟人工单层膜进行相互作用。经过拉膜的过程,再与未加载胞膜窖的单层膜作以对比,分析胞膜窖结构与单层膜之间的相互作用方式。直观的了解胞膜窖在单层膜上的分布与呈现形式。此部分研究采用实验研究和理论分析相结合的方法对DPPC/Chol二元混合体系在空气／水界面的相行为进行研究,具有较强的实际意义。通过以上两个方面的研究为胞膜窖结构的技术分离和功能探索提供了重要的实验依据和理论结果,具有较强的探索意义。
[Abstract]:Caveolae (Caveolae) on the cell membrane is approximately the size of diverse sag structure of 50-100nm, the signal protein involved in signal transduction are enriched in the exercise, a variety of biological functions, signal processing is the center of the cell, the main bearing inhibition of signal transduction. Caveolin-1 (Caveolin) for caveolae on specific marker protein, when the cell is in steady state or signal behavior guided by ligand, can be formed by scaffolding area (scaffold) fixed various signal molecules. As the biofilm structure of permanent membrane, the protein of physiological function of caveolae and cellar gradually attention, especially in signal transduction. Cholesterol transport, cell internalization and tumor suppression, muscle cell synthesis. The caveolar structure plays an important role in cell life activities, has important research value.
This paper studies the caveolar structure from two aspects: (1) purification and biological characterization of isolated caveolae structure; (2) the preliminary study of caveolae structure and in vitro simulated monolayer interaction. By studying these two aspects of the work, lay the foundation for further study on the structure and can as a function of caveolae, which is of great significance. The experimental results are as follows:
(1) and biological purification of caveolae structure: the experiment using 4 C Caveolae insoluble principle in nonionic detergent ThritonX-100 and the membrane area has unique characteristics of the flotage density, Caveolae. SDS-PAGE can be obtained by characteristic protein bands separated from the lung, the use of specific marker protein Caveolin-1 antibody detection point signal protein hybridization experiment, results showed that the Caveolin containing protein Caveolae. The protein content of samples obtained by Coomassie brilliant blue 0.79113mg/mL. of purified Caveolae were diluted to different concentration, obtained by atomic force microscopy Caveolae structure clear, the picture shows the size of the Caveolae 79 nm. final experimental results using the method of Caveolae and Caveolin protein were stable, good reproducibility, and can be applied to various biological tissues and cell lines A new method for fast separation of complete Caveolae and identification of Caveolae by dot blot was established.
(2) preliminary study of caveolae structure and in vitro simulated monolayer interactions: in order to study the composition of the cell membrane interaction mechanism between the pit structure of various membrane lipid components, we conducted simulation experiments in vitro. Through the LB membrane apparatus, the average sub area using surface pressure measurement (n -A) atomic force microscope and the observation curve, studied two palmitoyl phosphatidylcholine (DPPC) and cholesterol (Cholesterol) monolayer two mixture in the thermodynamic characteristics of AFM and the morphological characteristics of the air / water interface. PI -A curve analysis results show that when the temperature controlled at 20 DEG C (+ 0.5), mole fraction DPPC (XDPPC) is constant, with the increase of the surface pressure (PI), two mixed system area (A) decreased; when the surface pressure (PI) is constant, with the mole fraction of DPPC (XDPPC) increase in the membrane pressure of 15mN/m, two yuan hybrid system The area (A) has decreased first and then increased; but in the membrane pressure of 20mN/m, 25mN/m, 30mN/m, two yuan area of hybrid systems (A) presented a periodic nonlinear decrease; by Gibbs energy analysis, when XDPPC=0.7, the mixed system appeared maximum negative deviation, when the film pressure at 25mN/m, 30mN/m, DPPC/Chol two system appeared positive deviation trend in the XDPPC=0.1 theory are given. The system characteristics of.AFM observation interaction changes with PI and XDPPC also gives direct evidence. Finally, the caveolar structure we choose different ratio conditions and purification of artificial monolayer membrane interaction. Through the film, and not loaded monolayers of caveolae in contrast, analysis of the interaction between caveolae structure and monolayer. Understanding the distribution of caveolae in monolayer on the intuitive and presentation form Type. This part of research by using the method of experimental research and theoretical analysis of a combination of two mixture DPPC/Chol were studied in the phase behavior of the air / water interface, has a strong practical significance. Through the study of the above two aspects of technology and function for the separation of caveolae structure provides an important exploration
The experimental basis and theoretical results are of great significance for exploration.

【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329

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