酪氨酸酶催化交联丝素蛋白材料的研究
发布时间：2018-08-17 14:51
【摘要】：家蚕丝素蛋白具有优良的生物相容性，其用作组织工程支架等生物医用材料前景广阔。但未经交联的丝素材料易溶于水，难以实际应用。寻求一种使丝素蛋白材料的生物相容性和物理性能都较好，且可调控其生物降解速率的新的交联手段，是丝蛋白生物材料领域的迫切需求。酪氨酸酶是一种含铜的金属酶，，广泛分布于微生物、动植物及人体中，与生物体合成色素直接相关。本文用酪氨酸酶催化丝素大分子间的交联反应，制备再生丝素蛋白材料，对交联丝素材料的结构、物理性能以及体外酶降解行为进行系统性的研究，并以丝素膜为载体进行L929细胞体外培养，研究交联丝素膜的细胞相容性。 首先，用流延法制备酪氨酸酶催化交联的丝素膜。热水溶失率的测试结果表明交联后丝素膜的耐水性得到显著提高，膜的交联度随着酪氨酸酶添加量的增加而有所增大。交联膜的红外吸收光谱显示，在酪氨酸酶的催化作用下，丝素蛋白的酪氨酸残基氧化为邻苯醌，并与自由氨基发生分子间和分子内的交联。氨基酸分析结果表明，经交联后膜内酪氨酸和赖氨酸含量减少，进一步说明酪氨酸酶能够催化丝素蛋白中的酪氨酸和赖氨酸发生相互作用从而有效地交联丝素蛋白。X-射线衍射结果表明交联丝素膜仍以无定形结构为主，与未经交联的丝素膜相比，凝聚态无显著变化。丝素膜经酪氨酸酶催化交联后，断裂强度有所提高，断裂伸长率变化不明显。 其次，用冷冻干燥法制备酪氨酸酶催化交联的多孔材料。研究表明，经酪氨酸酶催化交联后，丝素多孔材料的热水溶失率明显降低。红外吸收光谱、X-射线衍射和热分析结果表明，交联后的多孔材料以无定形结构为主。交联多孔材料的孔径、孔隙率随着丝素溶液浓度的增大和冷冻温度的降低而减小。 再次，选择胶原酶IA作为体外催化丝素蛋白材料水解的模型酶，研究不同交联度的丝素膜的体外生物降解行为。结果表明，随着丝素膜交联度的增大，其降解速率显著减缓，降解产物中的游离氨基酸含量减少。通过调节交联度，可以调控丝素膜的生物降解速率。 最后，通过体外细胞培养研究了L929细胞在酪氨酸酶催化交联丝素膜上的粘附、生长和增殖状态。用荧光倒置显微镜观察了细胞生长状态，测定细胞黏附率，使用MTT比色法和考马斯亮蓝染色法测定细胞活力。结果表明交联丝素膜能够支持细胞的黏附、生长和增殖，酪氨酸酶催化交联膜与乙醇处理的纯丝素膜上的细胞活力无显著性差异。 通过本文的研究，为制备物理性能和生物相容性较好，且可调控其生物降解速率的丝素蛋白材料，提供了一种新的交联手段。
[Abstract]:Silk fibroin protein has good biocompatibility and is widely used as biomedical materials such as tissue engineering scaffold. But the uncrosslinked silk fibroin material is soluble in water and difficult to be applied in practice. It is an urgent need in the field of silk protein biomaterials to seek a new cross-linking method which can make silk fibroin materials have good biocompatibility and physical properties and can regulate their biodegradation rate. Tyrosinase is a copper-containing metalloenzyme widely distributed in microorganisms animals and plants and human body and directly related to biosynthetic pigments. In this paper, regenerated fibroin materials were prepared by using tyrosinase to catalyze cross-linking reaction between fibroin macromolecules. The structure, physical properties and enzymatic degradation behavior of cross-linked fibroin materials in vitro were systematically studied. L929 cells were cultured with silk fibroin membrane in vitro to study the cytocompatibility of cross-linked fibroin membrane. Firstly, the fibroin membrane catalyzed by tyrosinase was prepared by casting method. The results of hot water solubilization test showed that the water resistance of silk fibroin membrane increased significantly after crosslinking, and the crosslinking degree of silk fibroin membrane increased with the increase of tyrosinase content. The IR absorption spectra of the cross-linked membrane showed that the tyrosine residue of silk fibroin was oxidized to o-benzoquinone under the catalysis of tyrosinase and cross-linked with free amino groups both intramolecular and intramolecular. The results of amino acid analysis showed that the contents of tyrosine and lysine decreased after crosslinking. It is further demonstrated that tyrosinase can catalyze the interaction between tyrosine and lysine in fibroin, thus effectively crosslinking fibroin. X- ray diffraction results indicate that the cross-linked fibroin membrane is still amorphous. Compared with the uncrosslinked fibroin membrane, there was no significant change in the condensed state. When the fibroin membrane was crosslinked with tyrosinase, the breaking strength increased and the elongation at break did not change obviously. Secondly, the porous materials catalyzed by tyrosinase were prepared by freeze-drying method. The results showed that the hot water solution loss rate of fibroin porous materials decreased obviously after the crosslinking catalyzed by tyrosinase. The results of X-ray diffraction and thermal analysis show that the cross-linked porous materials are mainly amorphous. The pore size and porosity of crosslinked porous materials decrease with the increase of fibroin concentration and the decrease of freezing temperature. Thirdly, collagenase IA was selected as the model enzyme to catalyze the hydrolysis of silk fibroin in vitro, and the biodegradation behavior of silk fibroin membrane with different crosslinking degree was studied in vitro. The results showed that with the increase of the crosslinking degree of silk fibroin film, the degradation rate of silk fibroin film decreased significantly, and the content of free amino acid in the degradation product decreased. The biodegradation rate of silk fibroin membrane can be regulated by adjusting the crosslinking degree. Finally, the adhesion, growth and proliferation of L929 cells on the membrane catalyzed by tyrosinase were studied by cell culture in vitro. Cell growth was observed by fluorescence inverted microscope and cell adhesion was measured. Cell viability was determined by MTT colorimetry and Coomassie brilliant blue staining. The results showed that the cross-linked fibroin membrane could support cell adhesion, growth and proliferation. There was no significant difference in cell viability between the cross-linked membrane catalyzed by tyrosinase and the pure fibroin membrane treated with ethanol. This study provides a new cross-linking method for the preparation of silk fibroin material with good physical properties and biocompatibility and which can regulate its biodegradation rate.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
本文编号：2187992
[Abstract]:Silk fibroin protein has good biocompatibility and is widely used as biomedical materials such as tissue engineering scaffold. But the uncrosslinked silk fibroin material is soluble in water and difficult to be applied in practice. It is an urgent need in the field of silk protein biomaterials to seek a new cross-linking method which can make silk fibroin materials have good biocompatibility and physical properties and can regulate their biodegradation rate. Tyrosinase is a copper-containing metalloenzyme widely distributed in microorganisms animals and plants and human body and directly related to biosynthetic pigments. In this paper, regenerated fibroin materials were prepared by using tyrosinase to catalyze cross-linking reaction between fibroin macromolecules. The structure, physical properties and enzymatic degradation behavior of cross-linked fibroin materials in vitro were systematically studied. L929 cells were cultured with silk fibroin membrane in vitro to study the cytocompatibility of cross-linked fibroin membrane. Firstly, the fibroin membrane catalyzed by tyrosinase was prepared by casting method. The results of hot water solubilization test showed that the water resistance of silk fibroin membrane increased significantly after crosslinking, and the crosslinking degree of silk fibroin membrane increased with the increase of tyrosinase content. The IR absorption spectra of the cross-linked membrane showed that the tyrosine residue of silk fibroin was oxidized to o-benzoquinone under the catalysis of tyrosinase and cross-linked with free amino groups both intramolecular and intramolecular. The results of amino acid analysis showed that the contents of tyrosine and lysine decreased after crosslinking. It is further demonstrated that tyrosinase can catalyze the interaction between tyrosine and lysine in fibroin, thus effectively crosslinking fibroin. X- ray diffraction results indicate that the cross-linked fibroin membrane is still amorphous. Compared with the uncrosslinked fibroin membrane, there was no significant change in the condensed state. When the fibroin membrane was crosslinked with tyrosinase, the breaking strength increased and the elongation at break did not change obviously. Secondly, the porous materials catalyzed by tyrosinase were prepared by freeze-drying method. The results showed that the hot water solution loss rate of fibroin porous materials decreased obviously after the crosslinking catalyzed by tyrosinase. The results of X-ray diffraction and thermal analysis show that the cross-linked porous materials are mainly amorphous. The pore size and porosity of crosslinked porous materials decrease with the increase of fibroin concentration and the decrease of freezing temperature. Thirdly, collagenase IA was selected as the model enzyme to catalyze the hydrolysis of silk fibroin in vitro, and the biodegradation behavior of silk fibroin membrane with different crosslinking degree was studied in vitro. The results showed that with the increase of the crosslinking degree of silk fibroin film, the degradation rate of silk fibroin film decreased significantly, and the content of free amino acid in the degradation product decreased. The biodegradation rate of silk fibroin membrane can be regulated by adjusting the crosslinking degree. Finally, the adhesion, growth and proliferation of L929 cells on the membrane catalyzed by tyrosinase were studied by cell culture in vitro. Cell growth was observed by fluorescence inverted microscope and cell adhesion was measured. Cell viability was determined by MTT colorimetry and Coomassie brilliant blue staining. The results showed that the cross-linked fibroin membrane could support cell adhesion, growth and proliferation. There was no significant difference in cell viability between the cross-linked membrane catalyzed by tyrosinase and the pure fibroin membrane treated with ethanol. This study provides a new cross-linking method for the preparation of silk fibroin material with good physical properties and biocompatibility and which can regulate its biodegradation rate.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R318.08
【参考文献】
中国期刊全文数据库 前10条
1 吴徵宇,金宗明,徐力群;丝素的结晶度和结构变化的研究[J];蚕业科学;1993年02期
2 叶勇;张剑韵;黄龙全;;甘油和戊二醛对丝素膜性能的改良效果[J];蚕业科学;2006年02期
3 李明忠,卢神州,李丛新,李锋,吴徵宇;复合丝素膜的制备[J];纺织学报;1998年06期
4 闵思佳,陈芳芳,吴豪翔;环氧化合物与丝素蛋白化学交联凝胶的结构[J];高等学校化学学报;2005年05期
5 卢神州,李明忠,刘洋,张可达;聚乙二醇缩水甘油醚对丝素蛋白膜的改性[J];高分子材料科学与工程;2003年01期
6 刘永成,邵正中,孙玉宇,于同隐;蚕丝蛋白的结构和功能[J];高分子通报;1998年03期
7 周文;陈新;邵正中;;红外和拉曼光谱用于对丝蛋白构象的研究[J];化学进展;2006年11期
8 卢神州,李明忠,康宁,吴徵宇;环氧交联剂在丝素膜制备中的应用[J];丝绸;2000年03期
9 李明忠,严灏景;再生丝素的结构及其生物医学应用[J];丝绸;2000年05期
10 韩龙龙,张幼珠;稀土对丝素膜结构及溶解性的作用研究[J];丝绸;2002年04期
中国硕士学位论文全文数据库 前1条
1 汪宜宇;京尼平交联丝素蛋白材料的研究[D];苏州大学;2010年
本文编号：2187992
本文链接：https://www.wllwen.com/yixuelunwen/swyx/2187992.html
