仿生材料的制备及其与蛋白质及细胞的相互作用

发布时间：2018-03-10 22:33

本文选题：仿生材料　切入点：类GAG聚合物　出处：《苏州大学》2017年博士论文　论文类型：学位论文

【摘要】：仿生材料学,是指从分子水平上研究天然物质的结构特点和构效关系,进而开发出类似或优于原天然物质功能的新型材料。仿生的策略广泛应用于生物材料领域,通过模拟生命体中各种生物物质(如蛋白质和细胞等)的分子结构,促进相关的生物反应,将有利于材料与生命体的融合及材料生物功能的实现。生物物质之间的相互作用大致可分为活性结合与惰性排斥两种,前者是实现生命体内各种生理活动的途径,后者是保证各种生物活性激活的特异性和准确性的前提。因此,生物材料的仿生策略也应包含惰性和活性两个方面,即,在惰性背景之上特异性激活目标生物学过程,这样才能保证其功能的有效性。而仿生的核心环节则是模拟生物物质的功能性分子结构,进而实现类似的生物相互作用。细胞是生物体基本的结构和功能单元,其功能的实现依赖于细胞膜特殊的分子构成。细胞膜的磷脂结构提供了细胞内外环境之间的惰性屏障,而磷脂双分子层中结合的膜蛋白和膜糖等分子则介导了细胞与周围环境之间的信息、物质和能量的交换。本论文从仿生的角度出发,通过分子设计,分别模拟细胞膜中对细胞分化起关键作用的糖胺聚糖(glycosaminoglycan, GAG)成分以及作为惰性屏障的磷脂分子结构,探索了这些仿生分子在诱导干细胞向神经方向分化以及抗蛋白吸附方面的功能性。具体包含以下两部分内容：1.仿细胞膜糖结构的GAG类似物的合成及其对干细胞向神经方向分化行为的影响。GAG是细胞膜表面主要的糖结构,能够结合细胞质基质中多种蛋白质,是细胞信息传递过程中的主要媒介,特别在调控干细胞行为方面有显著影响。然而,GAG分子结构的多样性与不可控制性限制了其在生物工程方面的广泛应用。通过分子设计,模拟关键的GAG功能性分子结构,制备结构精确可控的GAG类似物,则可以取代天然GAG,实现相应的生物学功能。GAG的功能性分子结构主要包含硫酸酯基团和糖环结构,因此本论文分别采用小分子合成及共聚的方式实现了这两种结构的有机结合,获得了GAG类似物。首先,对糖类分子p-环糊精(β-cyclodextrin, β-CD)进行磺化改性,制备GAG类似物。分别通过硫-溴点击反应与铜催化叠氮-端炔环加成反应在β-CD上修饰了磺酸基团,得到分子结构有所差异的磺化β-CD:β-CD-(S-SO3Na)7与β-CD-(N3-SO3Na)7。细胞实验结果表明,这两种GAG类似物加入细胞培养液中均可促进L929细胞及胚胎干细胞生长,与天然GAG肝素相比,细胞数量没有明显差别,说明具有良好的细胞相容性。在诱导干细胞向神经方向分化的过程中,β-CD-(S-SO3Na)7与β-CD-(N3-SO3Na)7表现出较肝素更高的促分化效率。培养14天时,β-CD-(S-SO3Na)7与β-CD-(N3-SO3Na)7促进干细胞向神经分化的效率分别为肝素的1.2倍与1.9倍,而未磺化的β-CD其效率仅为肝素的0.6倍,与空白对照组相比无显著差异。因此,通过对糖类分子p-CD的磺化改性,可模拟天然GAG分子在促干细胞分化方面的生物学功能。在此基础上,为了进一步模拟GAG的长链结构,并调节功能性组分的相对含量,提出了制备GAG类似物的新概念,即,将GAG分子中“磺酸基团”与“糖基团”单元进行拆分和重组。首先,分别合成含有“磺酸基团”与“糖基团”的单体,利用可逆加成-断裂链转移聚合(Reversible Addition-Fragmentation Chain Transfer Polymerization, RAFT)共聚合和方式将两种功能性单元进行组合。通过改变投料比,可以精确调控共聚物中“磺酸基团”与“糖基团”的相对含量。细胞实验结果表明,该GAG类似物具有良好的细胞相容性,加入干细胞培养液中能够促进干细胞增殖。在诱导干细胞向神经方向分化的过程中,GAG类似物的存在能够提高分化效率,提高的程度与共聚物的组分相关。当磺酸基团与糖基团比例接近1：1时,共聚物促干细胞向神经分化的效率最高,是肝素的3.1倍。因此,这种类GAG共聚物可以通过精确调控功能单元的组成,实现更优于天然GAG的生物学功能。2.仿细胞膜磷脂结构的两性离子聚合物表面的制备及其抗蛋白吸附性能研究。磷脂双分子层外壁的两性离子结构是赋予细胞膜惰性屏障功能的关键,因此利用两性离子分子修饰的材料表面具有良好的抗污性能,而发展简单普适的修饰方法则是使两性离子表面得以广泛应用的关键环节。本文将末端带有黏性分子3,4-二羟基苯基-L-丙氨酸(3,4-dihydroxyphenyl-L-alanine, DOPA)的两性离子聚合物修饰在材料表面,研究了其抗蛋白质吸附的功能。利用修饰了DOPA的原子转移自由基聚合物(AtomTransfer Radical Polymerization, ATRP)引发剂分别引发单体磺酸基甜菜碱(sulfobetaine, SB)和羧酸基甜菜碱(carboxybetaine, CB)的聚合,制备了具有不同分子量的聚磺酸甜菜碱聚合物(poly (sulfobetaine), pSB)与聚羧酸甜菜碱聚合物(poly (carboxybetaine), pCB),并将其修饰在聚二甲基硅氧烷弹性体(polydimethylsiloxane, PDMS)表面。蛋白质吸附结果表明,聚合物分子量在一定范围内的增加有利于提高相应聚合物改性表面的抗蛋白质吸附能力；增加聚合物末端的DOPA数量有利于获得更为致密的聚合物修饰层,从而进一步提高了改性表面的抗蛋白质吸附能力。此外,在聚合物接枝过程中加入多巴胺小分子,也有利于提高聚合物层的致密性,从而进一步降低蛋白质吸附量。本论文从仿生细胞膜的角度出发分别获得了具有生物活性的GAG类似物及具有生物惰性的两性离子分子修饰的材料表面。未来的努力方向应是在材料界面将两者有机结合,抑制非特异性反应的同时促进目标生物活性,实现更全面的仿生功能。
[Abstract]:Biomimetic materials science, refers to the structure characteristics of the natural substances at the molecular level and structure-activity relationship, and develop new materials similar to or better than the original natural material function. Biomimetic strategy is widely used in the field of biological materials, through the simulation of various biological substances in life (such as protein and cell molecular structure, etc.) promote the biological reaction related, will be conducive to the realization of material and material life of the fusion and biological function. The interaction between biological materials can be divided into two kinds of binding activity and inert exclusion, the former is the way to realize the life in a variety of physiological activities, which is a prerequisite to ensure the specificity and accuracy of activation of various biological activity the strategy of bionics. Therefore, biological materials should also include two aspects, namely inert and active, activation of target biological processes in an inert background on specific, so as to protect Validity of their function. And it is the core link of bionic simulation of functional molecular structure of biological materials, so as to realize the interaction of similar biological cells. The organism is the structure and function of the basic unit, to achieve its function depend on the specific cell membrane phospholipids. The molecular structure of cell membrane provides inert barrier between the cells of the internal and external environment, and the combination of the phospholipid bilayer membrane proteins and membrane sugar molecules mediate between the cell and the surrounding environment information, the exchange of material and energy. From the point of view of biomimetic, through molecular design, simulate glycosaminoglycan plays a key role in cell differentiation in cell membranes (glycosaminoglycan, GAG) as well as the composition of phospholipid molecular structure of inert barrier, explored these bionic molecules in inducing stem cells to neural differentiation and anti protein adsorption. Can. Includes the following two parts: the synthesis and its effect on neural stem cell differentiation to the direction of the behaviors of the.GAG analogues of GAG 1. cell membrane mimetic structure of sugar is sugar structure of cell membrane surface mainly, can be combined with a variety of proteins in the cytoplasmic matrix, the cell information transmission medium in the process, especially significant effects in the regulation of stem cell behavior. However, the diversity of the molecular structure of GAG and could not be controlled to limit its application in biological engineering. Through molecular design, simulation GAG functional molecular structure key, GAG analogs are prepared accurately controllable structure, can replace natural functional GAG. The molecular structure of biological functions of the corresponding.GAG mainly contains sulfate group and sugar ring structure, so this paper respectively using small molecule synthesis and copolymerization of the structure of these two kinds of organic With access to the GAG analogues. First of all, the carbohydrate molecules p- cyclodextrin (P -cyclodextrin, P -CD) were sulfonated, preparation of GAG analogues. By sulfur bromine click reaction with copper - catalyzed azide alkyne cycloaddition reaction of sulfonic acid groups in modified beta -CD, obtained by the sulfonation beta -CD: beta -CD- molecular structure difference (S-SO3Na) 7 and -CD- (N3-SO3Na) 7. beta cells. The experimental results show that these two kinds of GAG analogues were added into the culture medium can promote the growth of L929 cells and embryonic stem cells, compared with natural GAG heparin, there was no difference in the number of cells, that has good cell compatibility. The differentiation of neural stem cells in the direction of induction, beta 7 and beta -CD- (S-SO3Na) -CD- (N3-SO3Na) 7 showed heparin can promote the differentiation of higher efficiency. When cultured for 14 days, beta -CD- (S-SO3Na) 7 and -CD- (N3-SO3Na) beta 7 promotes stem cells to neural differentiation efficiency respectively. 1.2 times and 1.9 times of heparin, and non sulfonated beta -CD and its efficiency is only 0.6 times of heparin, and the blank control group had no significant difference. Therefore, through sulfonation of carbohydrate molecules modified p-CD, mimics the natural biological function of GAG molecule in promoting stem cell differentiation on the basis of this aspect. In order to further simulate the structure, long chain GAG, and adjust the relative content of functional components, puts forward a new concept, the preparation of GAG analogues, GAG molecule "sulfonic acid groups" and "sugar group" unit of split and reorganization. Firstly, monomers were synthesized containing sulfonic acid groups. With "sugar group", addition fragmentation chain transfer polymerization (Reversible Addition-Fragmentation Chain Transfer using a reversible Polymerization, RAFT) and the copolymerization of two functional units are combined. By changing the feed ratio, can accurately control the copolymer" The relative content of sulfonic acid groups "and" sugar group ". The cell experiment results show that the GAG analogs has a good biocompatibility, add dry cell culture medium can promote the proliferation of stem cells. The differentiation of neural stem cells in the direction of induction, GAG analogs can improve the differentiation efficiency, and the degree of the compositions of the copolymers increased. When sulfonic acid and sugar group ratio close to 1:1, copolymer of promotion of stem cell differentiation into neurons of the highest efficiency is 3.1 times of heparin. Therefore, this kind of GAG copolymer can be composed by precise control function unit, realize the biological function of.2. cell membrane mimetic structure of phospholipid is better than natural GAG is an amphoteric ion polymer surface preparation and anti protein adsorption properties of zwitterionic structure. The phospholipid bilayer wall is given the key to the cell membrane inert barrier function, because of this Has good anti fouling properties by surface materials of zwitterionic molecular modification, and the modified method is simple and universal development is the key factor of the zwitterionic surface has been widely used. This paper will end with sticky molecules 3,4- two hydroxy phenyl alanine -L- (3,4-dihydroxyphenyl-L-alanine, DOPA) of the zwitterionic polymer modified on the surface of the material, study the anti protein adsorption function. Using modified DOPA atom transfer radical polymer (AtomTransfer Radical, Polymerization, ATRP) initiator respectively Inimer sulfo betaine (sulfobetaine, SB) and carboxyl betaine (carboxybetaine, CB) polymerization, were prepared with different molecular weight poly (sulfobetaine polymer poly (sulfobetaine), pSB) and poly carboxylic acid betaine polymer (poly (carboxybetaine), pCB), and the modified poly two methyl silicone An elastomer (polydimethylsiloxane, PDMS). The surface protein adsorption results showed that the molecular weight of the polymer increases in a certain range can improve the surface modification of the corresponding polymer anti protein adsorption capacity; increase the number of the end of the polymer DOPA is beneficial to obtain a more compact polymer modified layer, so as to further improve the modified surface resistance protein adsorption ability. In addition, adding dopamine small molecules in the polymer grafting process, but also help to improve the density of the polymer layer, thus further reducing the amount of adsorbed protein. This paper from the perspective of bionic membrane were obtained GAG analogues with biological activity and surface material of zwitterionic molecular modification of biologically inert.. the directions for future work should be in the material interface combining the nonspecific reaction and the goal of promoting students The activity of the material to achieve a more comprehensive bionic function.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TB391

【相似文献】