【作者】 王征；

【导师】 刘万顺；

【作者基本信息】 中国海洋大学 ， 水生生物学， 2005， 硕士

【摘要】 皮肤创伤是指机械性致伤因子所造成的皮肤损伤。皮肤创伤后的组织修复,是医学中最古老的课题之一。随着交通、建筑等行业的快速发展,皮肤的外伤性创伤越来越多见,并且伤情越来越复杂,治疗起来更加困难,尤其是患者本身患有糖尿病、静脉曲张等疾病,其创面治疗更加棘手。一些抗生素、维生素、微量元素等制剂虽然被广泛应用,但基本停留在等待伤口自然愈合阶段,促进创面愈合十分有限。一些生长因子虽然有较强的促进创面愈合作用,但因种类繁多,配伍复杂,可能引起增生性瘢痕,甚至引起癌变,尚处于实验研究阶段。因此研究具有促进创面愈合作用的药物意义重大。 甲壳素是自然界可再生的一种聚乙酰氨基葡萄糖高分子多糖。壳聚糖是甲壳素脱乙酰基的产物,由于其资源丰富、天然无毒、无抗原性、生物相容性好、可生物降解且具有特殊的生物活性,近20多年来,对壳聚糖及其衍生物的性质、功能研究和应用开发已得到广泛的重视。研究应用范围涉及到工业、农业、生物工程、医药卫生、食品工业、生物医用材料、畜牧水产、化学化工等众多领域,在医学领域更加突出,已有较多报道。但甲壳素和壳聚糖均不能直接溶于水,极大地限制了应用。而壳聚糖C₆位羟基最活泼,易于进行化学修饰改性,通过羧甲基化改性后的羧甲基壳聚糖具有良好的水溶性,因此在医药、生物材料、化妆品等领域有着广泛的应用前景。 本实验通过盐酸降解法和氧化降解法将高分子量的壳聚糖降解成不同分子量梯度的壳聚糖,然后将其羧甲基化制备成相应的不同分子量的羧甲基壳聚糖,同时对不同分子量的壳聚糖和相应分子量的羧甲基壳聚糖的各项指标进行测定。 分别以金黄色葡萄球菌和大肠杆菌为指示菌,研究不同分子量羧甲基壳聚糖对指示菌的最低抑菌浓度及其抑菌曲线,比较了不同分子量羧甲基壳聚糖对革兰氏阳性菌和革兰氏阴性菌的抑菌活性。结果表明:不同分子量羧甲基壳聚糖对金黄色葡萄球菌和大肠杆菌均具有抑菌活性;活性随着分子量的降低和浓度增高而增强;对金黄色葡萄球菌的抑制效果强于大肠杆菌。分子量为3KD左右的羧甲基壳聚糖的金黄色葡萄球菌最低抑菌浓度为0.5%,大肠杆菌的最低抑菌浓度为1%。 在细胞水平上通过显微观察法和MTT细胞计数法研究了不同分子量的羧甲更多还原

【Abstract】 Skin wound means skin is hurted by mechanical injury factors. Tissues healing after skin wound is one of the oldest problems in medicine. With the developing of automachinism, skin trauma is more and more popular and the wound becames more and more complex, so it is harder to be treated, especially some patient with diabetes or varicosity. Although some antibiotics, vitamine and microelement preparation are abroad used, their promotive effects are very finitary. Some grow factors have more promotive effects on wound healing, but they are in experimental study phase, because they have much categories and complex matches, or may be cause scar even cancer. So there is important signification to study medicines which have promotive effects on wound healing.Chitin is a kind of polyacetamideglucose macromolecular amylase. Chitosan is the product which is derived by chitin. Because of its resource abundance, nature, non-toxic, non-antigenic specificity, biodegradable and biocompatibility, the study on the characters, functions and appliance of chitosan and its derivitives are wide regarded in recently twenty years. They have wide applications in many fields such as industy agriculture, biology engineering, medicine, food industy, biomedicial materials, aquatic product and chemical industy, especially in the field of medicine. But the applications of chitin and chitosan are limited because they both are not straight soluable. The hydroxyl of chitosan C₆ can be chemical transformed because of its liveness, so the carboxymethylchitosan can be derived by chitosan. Carboxymethylchitosan is soluable, so it shows wide appliance foreground in the field of medicine, cosmetic and biomaterial.In this essay, Chitosan with different molecular weight were prepared by acid degradation and oxidation degradation, CM-Chitosan with different molecular weight is synthesized from corresponding Chitosan. Their physical properties are studied.Staphylococcus aureus and Escherichia coli are used as direction bacteria, MIC method is applied to evaluate the different repress effects. Results demonstrated that CM-Chitosan with different molecular weight can repress the proliferation of Staphylococcus aureus and Escherichia coli. The effects of low molecular weight CM-Chitosan are stronger than high molecular weight CM-Chitosan and the high concentration has more repressive effects. The CM-Chitosan （Mn=3KD） had the strongest promotive effects. MIC of Staphylococcus aureus is 0.5%, MIC of Escherichia coli is 1%.Microscope observation and MTT method are applied to evaluate the different effects on the proliferation of skin fibroblasts and keratinocytes. Results demonstrated that CM-Chitosan with different molecular weight can promote the proliferation of skin fibroblasts and keratinocytes at l¹000ppm and the concentrations at 100 ppm had the strongest effects; The effects of low molecular weight CM-Chitosan are stronger than high molecular weight CM-Chitosan, the CM-Chitosan （Mn=3KD） had the strongest promotive effects on skin fibroblasts and keratinocytes, which had equivalent effects compared with bFGF and EGF.New Zealand rabbits are used to make wound animal model by scalpel shearing. Morphologic and histological sections are applied to study the different effects on wound healing. Results demonstrated that CM-Chitosan with different molecular weight can promote the healing of skin wound at 5% concentration, the CM-Chitosan （Mn=3KD） had the strongest promotive effects on skin wound healing, which ahead of control for five days.All these results indicated that the CM-Chitosan （Mn=3KD） had thestrongest repress effects on StaphyJococcus aureus and Escherichia coli; strongest promotive effects on skin fibroblasts and keratinocytes, which had equivalent effects compared with bFGF and EGF; strongest promotive effects on healing of wound animal model, which provide the theory basis for theoretics study on structure and function, appliance and exploitation of the chitosan and its deritives with low molecular weight.更多还原