天然多糖AFG水凝胶的制备及其在皮肤创面愈合中的应用

	天然多糖AFG水凝胶的制备及其在皮肤创面愈合中的应用
	蒋泽秀
导师	吴明一
关键词	蜗牛糖胺聚糖，明胶，水凝胶，炎症，伤口愈合 Snail Glycosaminoglycan, Gelatin, Hydrogel, Inflammation, Wound Healing
摘要	蜗牛分泌的粘液具有很强的粘附性能，主要有机成分为蛋白质和糖胺聚糖 (Glycosaminoglycan, GAG)，其化学组成类似于细胞外基质。本课题组前期从白玉蜗牛粘液中分离出一种结构新颖的白玉蜗牛糖胺聚糖（Achatina fulica GAG，AFG），发现其具有结合炎症趋化蛋白和促进糖尿病小鼠皮肤伤口愈合的作用。然而，单独的糖胺聚糖应用于急性和慢性伤口时存在用药不便，给药量大而且需要持续给药等缺点。本文采用光催化AFG与甲基丙烯酰化明胶(Gelatin Methacryloyl, GelMA)共价交联成水凝胶，该水凝胶具有良好的生物相容性和可降解性，一次原位给药后即可实现促进创面愈合的药效。论文以肝素（Heparin，HEP）和透明质酸（Hyaluronic acid，HA）两种GAG作为对照，制备GAG与GelMA复合水凝胶。在成胶反应中，GAG作为生物活性物质和水凝胶体系骨架，其侧链羧基经EDC活化，与光引发自由基交联的GelMA反应，形成酰胺键和碳碳键等共价键交联的水凝胶。同时，本研究还对两个取代率的GelMA（G30和G100）、GelMA不同含量、不同类型GAG含量等影响成胶性能的参数进行了考察，开展水凝胶的流变性能、形貌、成胶时间、组织的粘附性和渗透性、刚性、稳定性等评价。采用扫描电镜、红外、固体核磁以及与组织粘附等实验，观察GAG/GelMA水凝胶的微观结构，并验证了酰胺键和碳碳键是其成胶的主要化学键类型，酰胺键和离子键是其粘附组织表面的主要化学键形式。粘附力学性能测试表明，光交联的GAG/GelMA水凝胶可在皮肤、肌肉等生物组织上快速原位成胶，并且与组织形成极强的粘附力；AFG/G30水凝胶具有湿粘附性能，其组织粘附强度优于市售的纤维蛋白胶。异硫氰酸荧光素标记AFG制备水凝胶的组织渗透试验表明，与组织表面的离子键、氢键等非共价键的相互作用，进一步增强AFG/G30的组织粘附性。刚性测试显示AFG/G30与皮肤软组织具有更好的弹性应变。水凝胶的流变力学表征发现，其杨氏模量小于或更接近人体皮肤。稳定性和吸水溶胀实验表明，GAG/GelMA具有良好的稳定性和吸水性能，有利于吸附伤口组织中的高渗出液。对AFG/G30水凝胶的生物安全性和伤口愈合作用进行评价结果显示，合成水凝胶的原料和AFG/G30水凝胶均无细胞毒性，GAG/G30水凝胶的提取物和浸出液均无溶血活性，但是HEP/G30的提取液有显著抗凝活性，不适用于伤口部位；AFG/G30水凝胶皮下植入在14天降解完全，与伤口愈合周期相匹配，避免敷料更换带来的二次损伤。AFG/G30水凝胶在伤口部位光照原位成胶，其伤口愈合速度显著快于G30和生理盐水组，体现了伤口愈合效果。对AFG/G30水凝胶促伤口愈合的作用机制研究结果显示，水凝胶的活性成分GAG与促炎趋化蛋白白介素8（IL8）、白介素6（IL6）、白介素1β（IL1β）、血小板因子4（PF4）和干扰素诱导蛋白10（IP10）具有高亲和力，AFG/G30水凝胶的条件培养基能减少IL8趋化的中性粒细胞数量，减少AFG/G30组中促炎细胞因子的含量，并且能显著抑制脂多糖（LPS）激活的TLR4/NF-?B信号通路。受蜗牛粘液启发的AFG/G30水凝胶具有优异的生物相容性和可降解性，能够粘附组织并可随伤口大小进行用药调整，只需一次用药便可达到伤口愈合效果。AFG/G30水凝胶通过吸附促炎趋化蛋白，减少伤口部位炎症细胞的浸润和炎症细胞因子含量，抑制炎症信号通路激活，降低促炎因子的表达。因此，AFG/G30水凝胶在愈合皮肤伤口方面显示出较好的应用潜力。; The mucus secreted by snails has strong adhesion properties, and the main organic components are protein and glycosaminoglycan (GAG), which are similar to extracellular matrix. In our previously study, a novel glycosaminoglycan AFG from the cultured China white jade snail mucus was purified, which can bind to inflammatory chemoattractant protein and promote skin wound healing in diabetic mice. However, glycosaminoglycan alone has some disadvantages such as inconvenient administration, large dose and continuous administration when used in acute and chronic wounds. In this study, AFG was covalently cross-linked with gelatin methacryloyl (GelMA) by photocatalytic reaction to form a hydrogel, which has good biocompatibility and biodegradability, and the efficacy of promoting wound healing can be achieved after one in situ administration. In this research, two GAGs, heparin (HEP) and hyaluronic acid (HA), were used as controls to prepare GAG and GelMA composite hydrogels. In the gelation reaction, GAG acts as a bioactive substance and the framework of the hydrogel system, and its side chain carboxyl groups are activated by EDC and react with GelMA, which is cross-linked by light-induced free radicals, to form covalent cross-links such as amide bonds and carbon-carbon bonds. At the same time, this study also investigated the parameters affecting the gelation properties, such as GelMA with two substitution rates (G30 and G100), different contents of GelMA, and different types of GAG contents. The rheological properties, morphology, gelation time, tissue adhesion and permeability, rigidity and stability of the hydrogel are evaluated. The microstructure of GAG/GelMA hydrogels was observed by scanning electron microscopy, infrared, solid-state nuclear magnetic resonance and tissue adhesion experiments, and it was verified that amide bonds and carbon-carbon bonds were the main chemical bond types, amide bonds and ionic bonds are the main chemical bond form of adhesion to the surface of the tissue. Adhesion mechanical performance test shows that the photocrosslinked GAG/GelMA hydrogel can quickly form gel in situ on biological tissues such as skin and muscle, and form strong adhesion with the tissue; AFG/G30 hydrogel has wet adhesion performance, and its tissue adhesion strength is superior to that of commercially available fibrin glue. The tissue permeation test of the hydrogel prepared by fluorescein isothiocyanate- labeled AFG showed that the interaction with non-covalent bonds such as ion bond and hydrogen bond on the tissue surface further enhanced the tissue adhesion of AFG/G30. Rigidity test showed that AFG/G30 had better elastic strain with skin soft tissue. Rheological mechanical characterization of the hydrogel found that its Young's modulus was smaller or closer to that of human skin. Stability and water-swelling experiments showed that GAG/GelMA had good stability and water-absorbing properties, which was beneficial for adsorbing high exudate
语种	中文
	2022-05
学位授予单位	中国科学院大学
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/75164
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	蒋泽秀. 天然多糖AFG水凝胶的制备及其在皮肤创面愈合中的应用[D]. 中国科学院大学,2022.

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