本文選題：蛋白暈 + 兩親性聚合物��； 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：隨著納米技術(shù)的興起,納米材料已經(jīng)廣泛應(yīng)用于各種領(lǐng)域,尤其是生物醫(yī)藥領(lǐng)域中。眾所周知,蛋白會與納米材料表面結(jié)合形成蛋白暈,這是一種不可避免的現(xiàn)象。經(jīng)過這些年的研究,人們對蛋白暈已經(jīng)有了初步的認(rèn)識。實際上蛋白暈就是納米材料作為外源物質(zhì)進(jìn)入生物體內(nèi)以后生物體產(chǎn)生免疫反應(yīng)的結(jié)果。蛋白的構(gòu)象改變可能會引起它功能的改變;納米材料的表面性質(zhì)和功能也會由于材料類型、尺寸、形狀、電荷、表面修飾等不同而表現(xiàn)出很大的差異。因此不同的納米材料與蛋白之間的相互作用會受到多種因素的綜合影響。有研究顯示蛋白吸附到納米材料表面上以后其構(gòu)象發(fā)生了改變。蛋白在受到納米材料影響的同時,也會影響納米材料。許多研究表明蛋白暈使納米材料在生物體內(nèi)的生物學(xué)特性、生物分布、細(xì)胞毒性等都發(fā)生了變化,這些變化會影響納米材料在生物體內(nèi)的功能,所以蛋白暈成為納米材料在生物醫(yī)藥領(lǐng)域應(yīng)用中的一大研究熱點。一般情況下,納米材料進(jìn)入體內(nèi)首先面對的是血液循環(huán)系統(tǒng),血漿中許多蛋白都能夠與納米材料形成蛋白暈,大多數(shù)研究也主要是聚焦在血漿蛋白與納米材料的相互作用上。本論文利用光學(xué)技術(shù)(吸收光譜、熒光光譜、動態(tài)光散射)、離心、尺寸排阻色譜、質(zhì)譜、凝膠電泳等方法研究了血漿中含量較高的蛋白(牛血清白蛋白、纖維蛋白原、轉(zhuǎn)鐵蛋白)與兩親性聚合物包被的金納米顆粒之間形成的蛋白暈,主要展開了以下工作:1.研究牛血清白蛋白與兩親性聚合物包被的金納米顆粒形成的離散型蛋白暈。用凝膠電泳的方法可以分離出吸附一個、兩個及多個牛血清蛋白的蛋白-納米顆粒復(fù)合物。一般情況下蛋白暈中所含蛋白的具體數(shù)目是很難計算的,通常都只是得到蛋白的相對含量。結(jié)合吸收光譜與離心法計算出納米顆粒表面飽和吸附蛋白的數(shù)目,結(jié)果說明兩親性聚合物包被的金納米顆粒表面能夠吸附的蛋白數(shù)目是有限的,這是由納米顆粒表面性質(zhì)和大小決定的。這種方法還可以計算不能形成離散型蛋白暈的蛋白(胰凝乳蛋白酶)在納米顆粒上的數(shù)目,最后推導(dǎo)出一個適用于兩親性聚合物包被的納米材料與蛋白形成的蛋白暈中蛋白數(shù)目的計算公式,為以后控制納米材料上吸附蛋白的數(shù)目打下基礎(chǔ)。2.測定牛血清白蛋白與兩親性聚合物包被的金納米顆粒形成的蛋白暈的尺寸。凝膠電泳、動態(tài)光散射、尺寸排阻色譜的原理都與被測分子的尺寸相關(guān),因此利用這三種方法能夠測定和計算牛血清白蛋白與形成的離散型蛋白暈的尺寸。之前分子模擬的結(jié)果已經(jīng)顯示牛血清白蛋白是嵌入到兩親性聚合物中的,所以兩親性聚合物包被的金納米顆粒吸附了蛋白之后尺寸并不會出現(xiàn)明顯的改變。對比三種尺寸計算方法得出的結(jié)果,最準(zhǔn)確的是尺寸排阻色譜法,其次是動態(tài)光散射,誤差最大的是凝膠電泳。但每種方法都有各自適用的體系,還是需要根據(jù)實際情況來選擇合適的計算方法。3.研究牛血清白蛋白與兩親性聚合物包被的金納米顆粒之間的相互作用力。之前的一系列結(jié)果都表明牛血清白蛋白與兩親性聚合物包被的金納米顆粒形成的蛋白暈非常穩(wěn)定。利用凝膠電泳和質(zhì)譜得到了牛血清白蛋白中三條與納米顆粒結(jié)合的多肽片段,長度分別是13、13和15個氨基酸殘基,每條片段都含有7個疏水性氨基酸。而聚丙烯酰胺凝膠電泳的結(jié)果顯示結(jié)合位點的多肽片段分子量約為13 kDa,再通過與牛血清白蛋白全部氨基酸序列的比對可以推斷出這條多肽片段全長應(yīng)該是105個氨基酸殘基,其中一共有46個疏水性氨基酸殘基,并且這些疏水性氨基酸分布比較集中,推測這段多肽的疏水性較強。熒光淬滅法也計算出牛血清白蛋白與兩親性聚合物包被的金納米顆粒之間的結(jié)合常數(shù)遠(yuǎn)大于一般的極限值,溫度變化與結(jié)合常數(shù)呈負(fù)相關(guān),說明二者的熒光淬滅模式是靜態(tài)淬滅,整個反應(yīng)體系的熱力學(xué)參數(shù)符合熱力學(xué)第二定律。這些數(shù)據(jù)都證明了它們之間的相互作用力非常強。吸附的驅(qū)動力有很多,但多肽片段的疏水性和兩親性聚合物的疏水鏈導(dǎo)致了二者吸附的主要驅(qū)動力是疏水作用力。4.研究轉(zhuǎn)鐵蛋白與兩親性聚合物包被的金納米顆粒形成的蛋白暈。同樣利用凝膠電泳、熒光淬滅法對轉(zhuǎn)鐵蛋白與兩親性聚合物包被的金納米顆粒形成的蛋白暈進(jìn)行了分析。轉(zhuǎn)鐵蛋白的分子量約77kDa,粒徑約為3.91nm,與牛血清白蛋白類似,所以轉(zhuǎn)鐵蛋白得到了與牛血清白蛋白相似的凝膠電泳結(jié)果。同時也發(fā)現(xiàn)轉(zhuǎn)鐵蛋白能夠與納米顆粒形成離散型蛋白暈。結(jié)合常數(shù)的大小說明它們之間的結(jié)合是非常穩(wěn)定的。轉(zhuǎn)鐵蛋白是癌癥治療中常用的靶向分子,因此研究它與納米顆粒形成的蛋白暈對于它的靶向應(yīng)用具有指導(dǎo)意義。5.研究纖維蛋白原與兩親性聚合物包被的金納米顆粒形成的蛋白暈。纖維蛋白原也屬于血漿中的含量較高的蛋白,纖維蛋白構(gòu)象的改變與一些通路的激活有關(guān)。因為纖維蛋白原的分子量和尺寸都非常大,所以它與兩親性聚合物包被的金納米顆粒形成的蛋白暈明顯較牛血清白蛋白和轉(zhuǎn)鐵蛋白大,在凝膠電泳上有明顯的滯后。熒光淬滅的數(shù)據(jù)也顯示纖維蛋白原與納米顆粒形成的是硬蛋白暈。納米顆粒結(jié)合一個如此大的分子肯定會對納米顆粒產(chǎn)生影響,今后還會繼續(xù)研究。
[Abstract]:With the rise of nanotechnology, nanomaterials have been widely used in various fields, especially in the field of biomedicine. It is well known that protein can be combined with the surface of nanomaterials to form a protein halo. This is an inevitable phenomenon. After these years, people have already had a preliminary understanding of the halo. In fact, the protein halo is the same. Nanomaterials are the result of the immune response of the organism after entering the organism. The conformation change of the protein may cause the change of its function; the surface properties and functions of the nanomaterials also show great differences due to the material type, size, shape, charge, surface modification and so on. The interaction between materials and proteins will be influenced by a variety of factors. Studies have shown that proteins are adsorbed on the surface of nanomaterials to change their conformations. The proteins can also affect nanomaterials while they are affected by nanomaterials. Many studies show that the protein halo makes the nanomaterials biological in the organism, Biological distribution and cytotoxicity have changed, and these changes will affect the function of nanomaterials in the organism. So the protein halo becomes a hot research hotspot in the application of biological medicine. In general, the nano materials are first faced with the blood circulation system, and many proteins in the plasma can be found. Most of the research is mainly focused on the interaction between plasma proteins and nanomaterials. This paper uses optical techniques (absorption spectra, fluorescence spectra, dynamic light scattering), centrifugation, size exclusion chromatography, mass spectrometry, gel electrophoresis and other methods to study the protein (bovine serum albumin) in plasma. Fibrinogen, transferrin, and the protein halo formed between two amphiphilic polymer coated gold nanoparticles. The following work is carried out mainly: 1. to study the discrete protein halo formed by the gold nanoparticles of bovine serum albumin and two amphiphilic polymer clad. A gel electrophoresis method can be used to separate one, two and multiple bovine blood. Albumin nanoparticles complex. In general, the specific number of proteins contained in the protein halo is difficult to calculate. Usually, the relative content of the protein is obtained. The number of the surface saturated adsorbed proteins on the surface of the nanoparticles is calculated by the absorption spectrum and centrifugation. The results indicate that the gold nanoparticles of the two amphiphilic polymer clad are shown. The number of proteins that can be adsorbed on the surface is limited, depending on the surface properties and size of the nanoparticles. This method can also calculate the number of proteins that can not form a discrete type of protein halo (chymotrypsin) on the nanoparticles. Finally, it derives an egg that is suitable for the formation of the two amphiphilic polymer coated nano materials and protein. The calculation formula of the number of protein in the white halo to determine the size of the protein halo formed by the determination of the number of adsorbed proteins on the nanomaterials by.2.. The principle of gel electrophoresis, dynamic light scattering, and size exclusion chromatography are all related to the size of the molecules. These three methods can determine and calculate the size of the BSA and the formed discrete protein halo. The results of the previous molecular simulation have shown that the bovine serum albumin is embedded in the two amphiphilic polymer, so the size of the gold nanoparticles covered by the two amphiphilic polymer clad is not significantly changed after the egg white. Compared to the results obtained by three methods of size calculation, the most accurate is size exclusion chromatography, followed by dynamic light scattering, and the maximum error is gel electrophoresis. But each method has its own system, or it is necessary to choose the appropriate method based on the actual situation,.3., to study the bovine serum albumin and the two amphiphilic polymer package. The interaction between gold nanoparticles. A series of previous results showed that the halo of bovine serum albumin and the gold nanoparticles of the two amphiphilic polymer coated gold nanoparticles were very stable. By gel electrophoresis and mass spectrometry, three peptide fragments of bovine serum albumin combined with nanoparticles were obtained, with the length of 13,13 and 15 ammonia, respectively. Each fragment contains 7 hydrophobic amino acids, and the results of polyacrylamide gel electrophoresis show that the molecular weight of the polypeptide fragment at the binding site is about 13 kDa, and the total length of this polypeptide fragment should be 105 amino acid residues by comparison with all the amino acid sequences of bovine serum albumin, with a total of 46 of them. The hydrophobic amino acid residues, and the distribution of these hydrophobic amino acids are concentrated, speculate that the polypeptide has a strong hydrophobicity. The fluorescence quenching method also calculated that the binding constant between the bovine serum albumin and the gold nanoparticles of the two amphiphilic polymer package is far greater than the general limit value, and the temperature change is negatively correlated with the binding constant. The fluorescence quenching mode of the two is statically quenched and the thermodynamic parameters of the whole reaction system conform to the second law of thermodynamics. These data prove that the interaction force between them is very strong. There are many driving forces of the adsorption, but the hydrophobicity of the polypeptide fragment and the hydrophobic chain of the two parent polymer lead to the main driving force of the two adsorption. It is a hydrophobic interaction force.4. to study the protein halo formed by the gold nanoparticles of the transferrin and two amphiphilic polymer coated gold nanoparticles. The gel electrophoresis and fluorescence quenching method are used to analyze the halo of the gold nanoparticles formed by the transferrin and the two amphiphilic polymer coated gold nanoparticles. The molecular weight of the transferrin is about 77kDa, the particle size is about 3.91nm, and the bovine blood. The albumin is similar, so the transferrin obtained the gel electrophoresis results similar to the bovine serum albumin. It also found that the transferrin could form a discrete protein halo with the nanoparticles. The binding constant of the binding constant indicates that the binding between them is very stable. The protein halo formed with the nanoparticles has a guiding significance for its targeting application.5. to study the protein halo formed by fibrinogen and gold nanoparticles containing two amphiphilic polymer packages. Fibrinogen is also a protein in the plasma, and the changes in the conformation of fibrin are related to the activation of some pathways. The molecular weight and size of the white source are very large, so the protein halo formed by the gold nanoparticles in the two amphiphilic polymer package is obviously larger than that of bovine serum albumin and transferrin, and there is a significant lag in gel electrophoresis. The fluorescence quenching data also show that the fibrinogen and nanoparticles form a hard protein halo. Such a large molecule will definitely affect the nanoparticles and will continue to study in the future.
【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R318.08

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本文編號：1949332