新型温度及糖响应性表面材料的制备及在细胞片层技术中的应用

发布时间：2019-01-30 09:06

【摘要】：智能材料因其对周围环境变化的灵敏、可控且可逆的响应性而成为研究热点。聚N-异丙基丙烯酰胺(PNIPAAm)是一种温度敏感性聚合物,目前已被广泛用于获取细胞片层,在无支架组织工程技术和再生医学领域展现出极大的潜力。然而,此种方法仍存在不足,如温度降低易引起细胞活性降低等问题,因此,开发新颖的智能材料成为迫切的需求。4-羧基间氟苯硼酸(CFPBA)和3-丙烯酰胺基苯硼酸(AAPBA)等苯硼酸衍生物可与葡萄糖或果糖等含有顺式二醇结构的生物小分子形成稳定的、可逆的共价作用,因而表现出可逆的糖响应性,在生物医药等领域有着广阔的应用前景。本文旨在制备具有温度或糖敏感性的聚合物,研究环境温度或糖浓度变化对其影响,并将其用作细胞培养基材,依据外界环境(如温度和糖浓度)的变化调控细胞或细胞片单层的贴附与脱附行为,藉此改进当前细胞片层的收获方式。首先,我们通过AGET-ATRP技术和后改性方式在玻璃基材表面制备出含有糖敏感性基团的聚合物分子刷,并通过糖敏感的可逆共价作用将端基修饰有生物活性粘附肽RGD、且侧链带有糖基的线型聚合物(RGD-PGAPMA)结合到玻璃表面,并用于细胞培养,通过在培养基中添加葡萄糖或果糖促使细胞自行脱附。将含糖培养基弃去并加入新鲜低糖普通培养基,可观察到细胞重新在基材表面贴附。上述研究表明,苯硼酸与顺式二醇的多重可逆共价作用可用于构建稳定、动态的生物界面材料,可通过改变培养基中糖浓度的方式实现表面生物活性“开-关”(on-off)作用,最终达到通过生物内刺激响应性(即生物体内糖浓度的变化)来调控细胞贴附与脱附行为的目的。其次,通过氧化还原聚合制备得到温度及糖双重响应性水凝胶片层。平衡溶胀率及表面亲疏水性质研究发现,温度降低或体系含糖时水凝胶膨胀且表面呈亲水性,即水凝胶表现出温度和糖的双重响应性。细胞实验表明,降低温度或更换含高糖培养基可以加快细胞片单层自行脱附,并得到完整的细胞片单层。并且,同时降低温度和更换高糖浓度的培养基时,细胞片层脱附速率最快。将已脱附的细胞片层重新在低糖普通培养基中培养,发现细胞仍保持较高活性。因此,温度及糖双重响应性水凝胶的使用加速了细胞片层的获取过程,并能使细胞保持较高的活性,对于细胞片层技术的推广应用具有重要意义。总之,本课题基于智能材料在细胞片层领域的研究基础,构建出新型的糖敏感性或温度及糖双重响应性基材,并成功用于细胞控释和细胞片层的获取,极大地丰富了细胞片层技术。
[Abstract]:Smart materials have become a research hotspot for their sensitive, controllable and reversible response to environmental changes. Poly (N-isopropylacrylamide) (PNIPAAm) is a kind of temperature-sensitive polymer, which has been widely used to obtain cell lamellae, and has shown great potential in the field of scaffolding tissue engineering and regenerative medicine. However, there are still some shortcomings in this method, such as the decrease of temperature will lead to the decrease of cell activity, so, The development of novel intelligent materials becomes an urgent need. 4-carboxyl m-fluorophenoboric acid (CFPBA) and 3-acrylamide phenylboric acid (AAPBA) can be combined with glucose or fructose and other organisms with cis-diol structure. Small molecules form stable, Because of its reversible covalent effect, it has a wide application prospect in biomedicine and other fields because of its reversible sugar response. The aim of this paper is to prepare polymers with temperature or sugar sensitivity, to study the effects of environmental temperature or sugar concentration on them, and to use them as substrate for cell culture. According to the change of environment (such as temperature and sugar concentration), the adhesion and desorption behavior of cell or cell slice monolayer can be regulated, so as to improve the current harvest mode of cell lamellae. Firstly, we prepared polymer brushes containing sugar sensitive groups on the surface of glass substrate by AGET-ATRP and post-modification, and modified the terminal groups with bioactive adhesion peptide RGD, by the reversible covalent reaction of sugar sensitivity. The side chain linear polymer (RGD-PGAPMA) with glycosyl group was bound to the glass surface and was used for cell culture. The cells were desorbed by adding glucose or fructose to the culture medium. After the sugar medium was discarded and added to the fresh low sugar medium, the cells were observed to be attached to the substrate surface again. The results show that the multiplex reversible covalent interaction of phenylboric acid and cis-diol can be used to construct stable and dynamic biomaterials. Surface biological activity (on-off) can be achieved by changing the concentration of sugar in the medium. Finally, the regulation of cell adhesion and desorption can be achieved through the intracellular stimulative response (i.e., the change of glucose concentration in the organism). Secondly, the temperature and sugar double responsive hydrogel lamellae were prepared by redox polymerization. The equilibrium swelling rate and surface hydrophobicity were studied. It was found that the hydrogel expanded and the surface was hydrophilic when the temperature decreased or the system contained sugar, that is, the hydrogel showed the dual response of temperature and sugar. Cell experiments showed that decreasing the temperature or replacing the medium containing high sugar could accelerate the desorption of the monolayer and obtain the complete monolayer of the cell piece. At the same time, the desorption rate of cell lamellae was the fastest when the temperature was lowered and the medium with high sugar concentration was replaced. The desorbed cell layer was recultured in low glucose medium, and the cells remained high activity. Therefore, the use of temperature and sugar double responsive hydrogels accelerates the process of obtaining cell lamellae and makes cells keep high activity, which is of great significance for the popularization and application of cell lamellar technology. In a word, based on the research of smart materials in cell lamellae, a novel sugar sensitivity or temperature and sugar dual responsive substrate was constructed, and it was successfully used for cell controlled release and cell lamellar acquisition. The technique of cell lamella is greatly enriched.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB381

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