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  本文選題：胞膜窖　切入點：去污劑法　出處：《陜西師范大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：胞膜窖(Caveolae)是存在于細胞膜表面大小約為50-100nm的多樣化凹陷結(jié)構(gòu),信號轉(zhuǎn)導(dǎo)過程所涉及的信號蛋白大多都富集于上,行使多種生物學(xué)功能,是細胞的信號處理中心,主要承載抑制信號轉(zhuǎn)導(dǎo)作用。窖蛋白(Caveolin)為胞膜窖上特異性標志蛋白,當(dāng)細胞處于穩(wěn)態(tài)或是由配體引導(dǎo)的信號行為時,可通過形成腳手架區(qū)域(scaffold)固定各種信號分子。作為質(zhì)膜上的永久的生物膜結(jié)構(gòu),胞膜窖及窖蛋白生理功能的研究逐漸受到人們的重視,尤其在信號轉(zhuǎn)導(dǎo)、膽固醇的運輸、細胞內(nèi)化及腫瘤抑制、肌細胞合成等方面。胞膜窖結(jié)構(gòu)在細胞生命活動過程中起著重要作用,具有重要的的研究價值。 本文對胞膜窖結(jié)構(gòu)從兩方面進行了研究：(1)胞膜窖結(jié)構(gòu)的分離純化及生物學(xué)鑒定；(2)胞膜窖結(jié)構(gòu)與體外人工模擬單層膜相互作用的初步研究。通過著手這兩方面的研究工作,可以為胞膜窖結(jié)構(gòu)及功能的進一步研究打下基礎(chǔ),具有重要意義。實驗結(jié)果如下： (1)胞膜窖結(jié)構(gòu)的分離純化及生物學(xué)鑒定：實驗利用4℃條件下Caveolae不溶于非離子去垢劑ThritonX-100以及所在膜區(qū)域具有獨特的浮力密度特性的原理,從豬肺中分離得到Caveolae。通過SDS-PAGE可得到特征蛋白條帶,利用特異性標志蛋白Caveolin-1抗體進行點雜交實驗檢測信號蛋白,結(jié)果表明Caveolae中含有Caveolin蛋白�？捡R斯亮藍法得到樣品蛋白含量為0.79113mg/mL。再對純化出的Caveolae作不同濃度的稀釋,通過原子力顯微鏡得到了清晰的Caveolae結(jié)構(gòu)圖,圖片結(jié)果顯示Caveolae的大小為79 nm。最終實驗結(jié)果表明用該種方法得到的Caveolae和Caveolin蛋白重顯性好、結(jié)果穩(wěn)定,可以應(yīng)用于各種生物組織及細胞系,建立了一種高效快速分離完整Caveolae并用點雜交鑒定Caveolae的新方法。 (2)胞膜窖結(jié)構(gòu)與體外人工模擬單層膜相互作用的初步研究：為了研究細胞膜上組成胞膜窖結(jié)構(gòu)各種脂質(zhì)組分之間的相互作用機理,我們進行了體外模擬試驗。通過LB膜儀,利用表面壓力-平均分子面積(π-A)曲線的測量和原子力顯微鏡觀測,研究了二棕櫚酰基磷脂酰膽堿(DPPC)和膽固醇(Cholesterol)二元混合體系單層膜在空氣/水界面上的熱力學(xué)特性及AFM形態(tài)學(xué)特征。π-A曲線分析結(jié)果表明,當(dāng)溫度控制在20(±0.5)℃,DPPC的摩爾分數(shù)(XDPPC)不變時,隨著表面壓力(π)的增加,二元混合系統(tǒng)的面積(A)不斷減��；當(dāng)表面壓力(π)不變時,隨著DPPC的摩爾分數(shù)(XDPPC)的增大,在膜壓為15mN/m時,二元混合系統(tǒng)的面積(A)出現(xiàn)了先減小后增大的趨勢；但在膜壓為20mN/m、25mN/m、30mN/m時,二元混合系統(tǒng)的面積(A)出現(xiàn)了非線性的周期性減��；通過吉布斯能量分析,當(dāng)XDPPC=0.7處,混合體系均出現(xiàn)了負偏差的極大值,當(dāng)膜壓在25mN/m、30mN/m時,DPPC/Chol二元體系在XDPPC=0.1出現(xiàn)正偏差趨勢。理論上給出了該體系相互作用隨π和XDPPC變化的特征。AFM的觀測也給出了直觀的證據(jù)。最后,我們選取不同比例條件下將純化出來的胞膜窖結(jié)構(gòu)與模擬人工單層膜進行相互作用。經(jīng)過拉膜的過程,再與未加載胞膜窖的單層膜作以對比,分析胞膜窖結(jié)構(gòu)與單層膜之間的相互作用方式。直觀的了解胞膜窖在單層膜上的分布與呈現(xiàn)形式。此部分研究采用實驗研究和理論分析相結(jié)合的方法對DPPC/Chol二元混合體系在空氣／水界面的相行為進行研究,具有較強的實際意義。通過以上兩個方面的研究為胞膜窖結(jié)構(gòu)的技術(shù)分離和功能探索提供了重要的 實驗依據(jù)和理論結(jié)果,具有較強的探索意義。
[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.

【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R329

【參考文獻】

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本文編號：1615184