基于纳米银的天然多糖水凝胶治疗糖尿病溃疡的研究

发布时间：2018-03-15 04:17

本文选题：糖尿病足溃疡　切入点：天然多糖　出处：《天津医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的近年来糖尿病发病率逐年攀升,长期血糖增高导致大血管、微血管受损并危及心、脑、肾、周围神经、眼睛、足等。糖尿病足溃疡作为糖尿病严重并发症之一,其病程反复、难愈合且有极高的致残率,给患者和家属带来极大的痛苦和沉重的经济负担。40%-80%的糖尿病足溃疡会合并感染,会加重溃疡治疗的难度。随抗生素的不断泛用和滥用,多重耐药菌的出现致使感染越来越难以控制。糖尿病足溃疡周围血液灌流受阻,抗菌药物在溃疡面的药物浓度低,难以达到抗感染的效果。针对这一难题,本文旨在合成一种抗菌且不诱导细菌耐药性的新型水凝胶伤口敷料,控制溃疡面细菌感染,促进伤口的愈合。方法将半胱氨酸接枝天然碱性多糖壳聚糖(CS-NAc)和马来酸苷接枝葡聚糖(Dex-Ma)加入纳米银(AgNPs)溶液中,构筑出三维网状结构的抗菌水凝胶。通过红外光谱(FT-IR)和核磁谱图分析材料结构,运用扫描电镜(SEM)观察抗菌水凝胶的微观结构,并通过溶胀测试来分析材料亲水性能,细胞毒性测试来评估材料的生物相容性,LIVE/DEAD细菌荧光染色测试用以评估材料抗菌性能。进一步以糖尿病大鼠全层皮肤缺损感染伤口为模型,观察材料对感染伤口的愈合作用,并取伤口处肉芽组织做组织形态学分析和免疫组织化学检测,分析巨噬细胞因子特异性抗原CD68和标记T细胞的因子CD3,评估伤口处的免疫能力及对炎症的防御能力。结果实验测定巯基化壳聚糖巯基含量和马来酸葡聚糖取代度,通过调节巯基与马来双键的摩尔比,在一定浓度AgNPs溶液条件下,制备含纳米银的天然多糖水凝胶(AgNPs@CNDM)水凝胶。扫描电镜图显示凝胶具有孔洞大小均一的三维网络结构;在5 min内,该复合凝胶溶胀率可达到400%,利用其溶胀性,可以迅速吸收组织渗液,有利于伤口愈合;细胞毒性测试结果显示,与AgNPs相比,细胞与AgNPs@CNDM水凝胶浸出液共培养48 h后,细胞存活率均大于80%,这可能是该复合凝胶能起到缓释Ag离子的作用,从而改善纳米银毒性,改善材料的安全性。抗菌实验结果表明,AgNPs@CNDM对金黄色葡萄球菌和铜绿假单孢菌均具有显著的抗菌效果。在糖尿病大鼠感染伤口模型实验中,AgNPs@CNDM组不仅可以有效地抑制感染还可以加速伤口的愈合,组织形态学进一步证实AgNPs@CNDM水凝胶对糖尿病大鼠感染伤口的抗炎促愈合作用。免疫组织化学检测表明,AgNPs@CNDM水凝胶可以提高CD68和CD3因子的表达,能在炎症早期阶段激活巨噬细胞,抑制炎症控制感染,提高糖尿病大鼠伤口的局部免疫能力,加速伤口愈合。结论本文通过简单、便捷方法制备出的三维结构天然多糖复合抗菌水凝胶,该凝胶具有较好的溶胀性能,可保持创面的湿性修复环境,且具有优良的生物相容性,并能有效地抑制细菌的增殖,对免疫因子CD68和CD3表达具有上调功能,促进感染伤口的愈合,且不诱导细菌耐药的产生,为临床治疗糖尿病足溃疡感染提供新策略和新思路。
[Abstract]:In recent years the incidence of diabetes is increasing year after year, long-term hyperglycemia leads to large blood vessels, microvascular damage and endanger the heart, brain, kidney, peripheral nerves, eyes, feet. Diabetic foot ulcer is one of the serious complications of diabetes mellitus, the duration of healing and relapse, with high morbidity, diabetic foot ulcer for patients and their families great pain and heavy economic burden of.40%-80% associated with infection, will increase the difficulty of treatment. With the continuous ulcer pan antibiotics use and abuse, resulting in the emergence of multiple drug resistant infection is becoming increasingly difficult to control diabetic foot ulcer. Peripheral blood perfusion is blocked, antibiotics in ulcer surface drug concentration is low, it is difficult to achieve anti the effect of infection. Aiming at this problem, this paper aims to synthesize a novel antibacterial hydrogel wound dressing without inducing bacterial resistance, the ulcer control bacterial infection, promote wound Healing. Methods the cysteine grafted natural alkaline polysaccharide chitosan (CS-NAc) and maleic acid grafted dextran (Dex-Ma) nano silver (AgNPs) solution, build a three-dimensional network structure. The antibacterial hydrogel by infrared spectroscopy (FT-IR) and NMR spectra analysis of the material structure, using scanning electron microscopy (SEM) microstructure observation of antibacterial hydrogel, and by swelling test to analyze the hydrophilic properties of materials, cell toxicity test to evaluate the biocompatibility of LIVE/DEAD bacterial staining testing to evaluate the antibacterial function of diabetic rats. Further to full-thickness skin defect wound infection model, observation materials on infected wound healing and wound at the granulation tissue for morphological analysis and immunohistochemistry analysis, factor CD3 factor macrophage specific antigen CD68 and T cell markers, the evaluation of the wound Immunity and inflammation on defense. Results the determination of thiolated chitosan sulfhydryl content and maleic acid dextran substitution, by regulating the molar ratio of thiol and maleic double bonds, in a certain concentration of AgNPs solution under natural conditions, polysaccharide gel preparation containing silver nanoparticles (AgNPs@CNDM) hydrogel. The SEM shows that the three-dimensional network structure gel holes with uniform size; within 5 min, the composite gel swelling rate can reach 400%, the swelling, can rapidly absorb tissue fluid, is conducive to wound healing; cell toxicity test results showed that compared with AgNPs cells and AgNPs@CNDM hydrogel extracts 48 h after co culture, cell survival rate more than 80%, it is possible that the composite gel can play the role of Ag + release, thereby improving the nano silver toxicity, improve the safety of antibacterial materials. The experimental results show that the AgNPs@CNDM of golden yellow Staphylococcus aureus and Pseudomonas aeruginosa had significant antibacterial effect in diabetic rats. The wound infection model experiment, AgNPs@CNDM group can not only effectively inhibit infection can accelerate wound healing, tissue morphology further confirmed that AgNPs@CNDM hydrogel promote healing of diabetic rats infected wounds anti-inflammatory. Immunohistochemistry showed that AgNPs@CNDM the hydrogel can improve the expression of CD68 and CD3 factor, can activate macrophages in the early stage of inflammation, inhibiting the inflammatory infection control, improve the local immunity wound in diabetic rats, accelerate wound healing. The conclusion of this paper through the simple and convenient preparation method of the three-dimensional structure of natural polysaccharide composite antibacterial gel, the gel has good swelling properties can keep wet, repair wound environment, and has excellent biocompatibility, and can effectively inhibit bacteria Proliferation plays an up-regulation role in the expression of CD68 and CD3, and promotes wound healing, and does not induce bacterial resistance. It provides new strategies and new ideas for clinical treatment of diabetic foot ulcer infection.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2

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