丝胶蛋白水凝胶的制备及应用研究

发布时间：2018-09-09 13:54

【摘要】：第一部分一种可注射且具有荧光特性和细胞粘附性的3D丝胶蛋白水凝胶应用于再生医学的研究目的：探索天然的丝胶蛋白在组织工程与再生医学中的潜在应用及价值。方法：我们采用LiBr方法从家蚕丝素缺失型家蚕品种(185ND-s)中分离丝胶蛋白。再将丝胶蛋白与交联剂戊二醛交联制备丝胶水凝胶。然后对丝胶蛋白水凝胶的理化特性和生物相容性以及缓释药物功能进行了系统的表征及研究。根据材料的特征探讨了该水凝胶在组织工程与再生医学中的潜在应用及价值。结果：我们从丝素缺失突变型蚕茧中成功分离出了天然结构未破坏的丝胶蛋白并制备了共价交联的三维丝胶蛋白水凝胶。该水凝胶具有可注射性,便于微创植入体内；具有荧光特性,可用于生物成像和体内跟踪;另外,该水凝胶具有优良的细胞粘附性,可以有效地促进细胞粘附、增殖和各种细胞的长期存活；丝胶蛋白水凝胶具有很好的控释生物因子的功能；水凝胶还拥有良好的弹性、高孔隙率和pH依赖降解特性。这些特性都是丝胶蛋白水凝胶作为细胞和药物载体的优势。结论：该丝胶蛋白水凝胶在组织工程与再生医学领域具有广泛的用途。第二部分具有荧光特性的丝胶-海藻酸盐互穿双网络水凝胶用于细胞和药物递送载体的研究目的：为了改善海藻酸盐水凝胶的性质,如机械强度、药物缓释能力和细胞粘附性等。方法：本研究引入了互穿网络的设计理念,采用天然的丝胶蛋白(从丝素缺失型家蚕蚕茧185Nd-s中分离而来)与海藻酸盐在交联剂戊二醛和氯化钙的同时作用下制备了互穿双网络结构水凝胶。采用扫描电子显微镜观察了冻干的水凝胶微观结构；水凝胶的孔隙率山液体置换方法得到；采用傅里叶红外光谱仪检测了丝胶蛋白二级结构的变化;水凝胶的机械性能由万能材料测试机测试；水凝胶的膨胀和降解由称重方法获得；药物缓释采用HRP作为模式药物进行评估：水凝胶的荧光和互穿双网络结构由荧光显微镜和激光共聚焦显微观察得到;水凝胶的体内跟踪实验由小动物活体成像系统实施；水凝胶的细胞粘附性和水凝胶促进细胞增殖、迁移和长期存活的实验由CCK8、观察和细胞计数统计等分析方法实现。结果：(1)我们成功地制备了丝胶蛋白-海藻酸盐互穿双网络结构水凝胶。(2)该复合水凝胶具有荧光特性,便于体内跟踪与检测。(3)水凝胶的弹性模量,降解和药物缓释动力学均可通过改变丝胶蛋白和海藻酸盐的比例在一定范围内进行调节。(4)复合水凝胶具有高孔隙率特征。(5)复合水凝胶具有良好的生物相容性,适于作为细胞外基质,可以长期支持细胞的存活、增殖与迁移。结论：本研究揭示了丝胶蛋白-海藻酸盐水凝胶可作为细胞或药物递送的多功能平台用于各种组织再生。本研究同时也为进一步探索丝胶用于和别的材料复合制备互穿网络结构改善相关材料的性质提供了一种设计理念。
[Abstract]:Part 1 A injectable 3D sericin hydrogel with fluorescent properties and cell adhesion for regenerative medicine
Objective: To explore the potential application and value of natural sericin in tissue engineering and regenerative medicine.
METHODS: Sericin was isolated from silkworm silkworm (185ND-s) by LiBr method. Sericin hydrogels were prepared by crosslinking sericin with glutaraldehyde. The physicochemical and biocompatibility of sericin hydrogels and the function of sustained-release drugs were characterized and studied. The potential application and value of the hydrogel in tissue engineering and regenerative medicine were discussed.
RESULTS: We successfully isolated unstructured sericin from silk fibroin-deficient mutant cocoons and prepared a covalently crosslinked three-dimensional sericin hydrogel. The hydrogel is injectable and easy to be implanted in vivo. It has fluorescent properties and can be used for bioimaging and in vivo tracking. Good cell adhesion can effectively promote cell adhesion, proliferation and long-term survival of various cells; sericin hydrogels have good function of controlling release of biological factors; hydrogels also have good elasticity, high porosity and pH-dependent degradation characteristics. These characteristics are sericin hydrogels as cell and drug carriers. The advantage.
Conclusion: the sericin hydrogel is widely used in tissue engineering and regenerative medicine.
Part II Sericin-alginate interpenetrating double-network hydrogels with fluorescent properties for cell and drug delivery
OBJECTIVE: To improve the properties of alginate hydrogels, such as mechanical strength, drug release and cell adhesion.
METHODS: Introducing the design concept of interpenetrating network (IPN), IPN hydrogels were prepared by using natural sericin protein (isolated from silk fibroin-deficient silkworm cocoon 185 Nd-s) and alginate in the presence of crosslinking agent glutaraldehyde and calcium chloride. The mechanical properties of the hydrogel were tested by universal material testing machine; the swelling and degradation of the hydrogel were obtained by weighing method; the sustained release of the drug was evaluated by HRP as a model drug. The fluorescence and interpenetrating double network structures of the hydrogels were observed by fluorescence microscopy and laser confocal microscopy; the in vivo tracking experiments of the hydrogels were performed by a small animal imaging system; the experiments of cell adhesion and hydrogels promoting cell proliferation, migration and long-term survival were analyzed by CCK8, observation and cell counting statistics. Method implementation.
Results: (1) We successfully prepared the sericin-alginate interpenetrating double-network hydrogel. (2) The composite hydrogel has fluorescence characteristics and is easy to track and detect in vivo. (3) The elastic modulus, degradation and drug release kinetics of the hydrogel can be adjusted by changing the ratio of sericin to alginate in a certain range. (5) Composite hydrogels have good biocompatibility and can be used as extracellular matrix to support cell survival, proliferation and migration for a long time.
CONCLUSION: This study reveals that sericin-alginate hydrogel can be used as a multifunctional platform for cell or drug delivery for tissue regeneration.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R318.08

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