丝素肽结构与止血性能的研究

发布时间：2018-06-21 11:58

本文选题：丝素肽 + 分子质量　；参考：《浙江理工大学》2015年硕士论文

【摘要】：日常生活中对突发性事故的急救治疗，医院里对病人手术过程中的创伤止血，战争中对受伤战士的现场救护，对于伤者局部有效的快速止血非常重要。未受控制的出血已经成为威胁伤员生命，导致突发事故、医疗手术或战场死亡的主要原因。快速有效的止血材料可大幅度降低伤员的死亡率。因此，开发快速、安全、有效的新型止血材料成为医学材料领域的重要研究课题。丝素蛋白作为从蚕丝中提取的一种天然高分子材料，具有无毒、炎症反应低、生物相容性良好、可生物降解等特点，广泛应用于药物控释、人造皮肤、人造韧带以及组织工程等领域。丝素蛋白的水解产物丝素肽，在日用化工、食品加工以及生物医药等领域均有开发应用，但丝素肽在止血材料的研究应用尚未见报道。现阶段对于粉状止血材料的研究较少，鉴于粉状止血材料使用及存储方便，对其深入的研究探讨很有必要。本文采用CaCl2-乙醇-水（摩尔比1:2:8）三元体系、磷酸和氢氧化钠对脱胶蚕丝进行水解，通过喷雾干燥和冷冻干燥制备不同分子量的丝素肽，对其结构和性能进行测试分析，并用于止血实验和体外细胞毒性实验，初步研究并探讨了丝素肽的理化性质和止血性能之间的关系，主要研究内容如下： 1.以CaCl2-乙醇-水（摩尔比1:2:8）三元体系为水解溶液，以58℃，68℃，78℃，88℃，98℃五档水解温度和1h，3h，9h，24h四种水解时间为不同条件水解脱胶蚕丝，通过喷雾干燥和冷冻干燥制备盐解丝素肽。采用凝胶过滤色谱（GPC）、傅里叶红外变换光谱（FTIR）、X-射线衍射（XRD）、扫描电镜（SEM）和氨基酸组分分析等测试手段对丝素肽的分子量分布、聚集态结构、表面形貌及氨基酸组成等进行表征。结果表明，随着水解温度的提高和水解时间的延长，丝素肽的分子量降低，丝素肽的聚集态结构以无规卷曲为主，存在少量的β-折叠结构，丝素肽粉末形貌以表面褶皱的球囊状颗粒为主，存在球形颗粒，粒径随着分子量的降低变小且更为均匀，主要氨基酸组分包括丝氨酸、甘氨酸、丙氨酸和酪氨酸四种，其组分及含量与水解温度和水解时间无明显关联； 2.以磷酸溶液为水解溶液，设计磷酸浓度、水解温度和水解时间三因素正交水解实验，通过冷冻干燥制备酸解丝素肽粉末。采用同样的测试手段对酸解丝素肽进行性能表征。结果表明，酸解丝素肽的分子量明显降低，主要分布在3.8~12.6kDa，丝素肽的聚集态结构为无规卷曲，丝素肽粉末呈现无规碎片状，主要氨基酸组分包括甘氨酸、丙氨酸、天冬氨酸和谷氨酸等。 3.以氢氧化钠溶液为水解溶液，设计氢氧化钠浓度、水解温度和水解时间三因素正交水解实验，通过冷冻干燥制备丝素肽粉末。测试手段同上所述。结果表明，碱解丝素肽的分子量较低，分布集中在3.6~18.9kDa，丝素肽的聚集态结构为无规卷曲，丝素肽粉末表面形貌与酸解丝素肽相似，，主要氨基酸组分为天冬氨酸、谷氨酸、丙氨酸和酪氨酸等。 4.不同水解方法得到的丝素肽的止血性能及体外细胞毒性研究。通过凝血弹性描记（TEG）测试、大鼠肝脏创伤止血模型和体外细胞毒性实验表征丝素肽的止血性能和生物相容性。结果表明，高分子量盐解丝素肽具备与Arista相近的止血性能，而低分子量盐解丝素肽、酸解丝素肽和碱解丝素肽则无显著的止血性能；各种水解方法制备的丝素肽对细胞的生长繁殖无毒副作用。
[Abstract]:The rapid , safe and effective hemostatic material is an important research topic in the field of medical materials .

Silk fibroin is a kind of natural high molecular material extracted from silk , and has the characteristics of no toxicity , low inflammation , good biocompatibility , biodegradability and the like , and is widely applied to the fields of drug controlled release , artificial skin , artificial ligament and tissue engineering .

In this paper , CaCl2 - ethanol - water ( molar ratio 1 : 2 : 8 ) ternary system , phosphoric acid and sodium hydroxide were used to hydrolyze the degumming silk . The structure and properties of silk fibroin peptides with different molecular weights were prepared by spray drying and freeze drying . The relationship between the physicochemical properties and hemostatic properties of silk fibroin peptide was studied . The main contents of the study were as follows :

The molecular weight distribution , aggregation state structure , surface morphology and amino acid composition of silk fibroin peptide were characterized by means of spray drying and freeze drying .

2 . The acid solution was prepared by orthogonal hydrolysis with phosphoric acid solution as hydrolysis solution , phosphoric acid concentration , hydrolysis temperature and hydrolysis time . The results showed that the molecular weight of the fibroin peptide was obviously reduced , the main distribution was 3.8 - 12.6 kDa , the aggregation state of silk fibroin peptide was random , and the main amino acid component included glycine , alanine , aspartic acid and glutamic acid .

3 . Using sodium hydroxide solution as hydrolysis solution , designing sodium hydroxide concentration , hydrolysis temperature and hydrolysis time three factors orthogonal hydrolysis experiment , and preparing silk fibroin powder by freeze - drying . The results showed that the molecular weight of silk fibroin peptide was lower , the distribution concentration was 3.6 - 18.9 kDa , the aggregation state of silk fibroin peptide was 3 . 6 - 18 . 9 kDa , the morphology of silk fibroin peptide was similar to that of acid - releasing silk peptide , and the main amino acid component was aspartic acid , glutamic acid , alanine and tyrosine .

4 . The hemostatic properties and in vitro cytotoxicity of silk fibroin peptides obtained by different methods of hydrolysis were studied . The hemostatic properties and biocompatibility of silk fibroin peptides were characterized by thromboelastography ( TEG ) test , rat liver trauma hemostasis model and in vitro cytotoxicity experiment .
The silk fibroin peptide prepared by various hydrolysis methods has no toxic or side effect on the growth and propagation of cells .
【学位授予单位】：浙江理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R612;TS106

【参考文献】