【摘要】：智能材料因其對周圍環(huán)境變化的靈敏、可控且可逆的響應(yīng)性而成為研究熱點。聚N-異丙基丙烯酰胺(PNIPAAm)是一種溫度敏感性聚合物,目前已被廣泛用于獲取細(xì)胞片層,在無支架組織工程技術(shù)和再生醫(yī)學(xué)領(lǐng)域展現(xiàn)出極大的潛力。然而,此種方法仍存在不足,如溫度降低易引起細(xì)胞活性降低等問題,因此,開發(fā)新穎的智能材料成為迫切的需求。4-羧基間氟苯硼酸(CFPBA)和3-丙烯酰胺基苯硼酸(AAPBA)等苯硼酸衍生物可與葡萄糖或果糖等含有順式二醇結(jié)構(gòu)的生物小分子形成穩(wěn)定的、可逆的共價作用,因而表現(xiàn)出可逆的糖響應(yīng)性,在生物醫(yī)藥等領(lǐng)域有著廣闊的應(yīng)用前景。本文旨在制備具有溫度或糖敏感性的聚合物,研究環(huán)境溫度或糖濃度變化對其影響,并將其用作細(xì)胞培養(yǎng)基材,依據(jù)外界環(huán)境(如溫度和糖濃度)的變化調(diào)控細(xì)胞或細(xì)胞片單層的貼附與脫附行為,藉此改進當(dāng)前細(xì)胞片層的收獲方式。首先,我們通過AGET-ATRP技術(shù)和后改性方式在玻璃基材表面制備出含有糖敏感性基團的聚合物分子刷,并通過糖敏感的可逆共價作用將端基修飾有生物活性粘附肽RGD、且側(cè)鏈帶有糖基的線型聚合物(RGD-PGAPMA)結(jié)合到玻璃表面,并用于細(xì)胞培養(yǎng),通過在培養(yǎng)基中添加葡萄糖或果糖促使細(xì)胞自行脫附。將含糖培養(yǎng)基棄去并加入新鮮低糖普通培養(yǎng)基,可觀察到細(xì)胞重新在基材表面貼附。上述研究表明,苯硼酸與順式二醇的多重可逆共價作用可用于構(gòu)建穩(wěn)定、動態(tài)的生物界面材料,可通過改變培養(yǎng)基中糖濃度的方式實現(xiàn)表面生物活性“開-關(guān)”(on-off)作用,最終達到通過生物內(nèi)刺激響應(yīng)性(即生物體內(nèi)糖濃度的變化)來調(diào)控細(xì)胞貼附與脫附行為的目的。其次,通過氧化還原聚合制備得到溫度及糖雙重響應(yīng)性水凝膠片層。平衡溶脹率及表面親疏水性質(zhì)研究發(fā)現(xiàn),溫度降低或體系含糖時水凝膠膨脹且表面呈親水性,即水凝膠表現(xiàn)出溫度和糖的雙重響應(yīng)性。細(xì)胞實驗表明,降低溫度或更換含高糖培養(yǎng)基可以加快細(xì)胞片單層自行脫附,并得到完整的細(xì)胞片單層。并且,同時降低溫度和更換高糖濃度的培養(yǎng)基時,細(xì)胞片層脫附速率最快。將已脫附的細(xì)胞片層重新在低糖普通培養(yǎng)基中培養(yǎng),發(fā)現(xiàn)細(xì)胞仍保持較高活性。因此,溫度及糖雙重響應(yīng)性水凝膠的使用加速了細(xì)胞片層的獲取過程,并能使細(xì)胞保持較高的活性,對于細(xì)胞片層技術(shù)的推廣應(yīng)用具有重要意義。總之,本課題基于智能材料在細(xì)胞片層領(lǐng)域的研究基礎(chǔ),構(gòu)建出新型的糖敏感性或溫度及糖雙重響應(yīng)性基材,并成功用于細(xì)胞控釋和細(xì)胞片層的獲取,極大地豐富了細(xì)胞片層技術(shù)。
[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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TB381

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