氧化铜纳米酶对糖尿病小鼠全层皮肤缺损创面修复的作用及其机制  

Effects and mechanism of copper oxide nanozymes on wound healing of full-thickness skin defects in diabetic mice

作　　者：彭源 卢毅飞 邓君 张艳[1] Peng Yuan;Lu Yifei;Deng Jun;Zhang Yan(Department of Plastic and Reconstructive Surgery,Shanghai Ninth People′s Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200011,China;State Key Laboratory of Trauma,Burns and Combined Injury,Institute of Burn Research,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing Key Laboratory for Disease Proteomics,Chongqing 400038,China)

机构地区：[1]上海交通大学医学院附属第九人民医院整复外科,200011 [2]陆军军医大学(第三军医大学)第一附属医院全军烧伤研究所,创伤、烧伤与复合伤国家重点实验室,重庆市疾病蛋白质组学重点实验室,400038

出　　处：《中华烧伤杂志》2020年第12期1139-1148,共10页Chinese Journal of Burns

基　　金：国家自然科学基金(81920108022);重庆市自然科学基金(cstc2020jcyj-msxmX1057)。

摘　　要：目的探讨氧化铜纳米酶对糖尿病小鼠全层皮肤缺损创面修复的作用及其机制。方法(1)采用氯化铜与L-抗坏血酸反应的方法合成氧化铜纳米酶。采用透射电子显微镜观察氧化铜纳米酶大小和形貌,动态光散射粒度分析仪和纳米粒度电位仪分别分析其水合粒径和表面电位。(2)采用过氧化氢检测试剂盒、超氧阴离子检测试剂盒、3,3′,5,5′-四甲基联苯胺显色剂分别测定150 ng/mL氧化铜纳米酶的过氧化氢、超氧阴离子、羟自由基清除能力,计算过氧化氢、超氧阴离子、羟自由基清除比例(样本数均为3)。(3)将小鼠成纤维细胞系3T3细胞采用随机数字表法(分组方法下同)分为空白对照组、单纯过氧化氢组和过氧化氢+氧化铜组,每组3孔。将终质量浓度25 ng/mL的氧化铜纳米酶加入过氧化氢+氧化铜组细胞中预处理30 min。然后,在单纯过氧化氢组和过氧化氢+氧化铜组细胞中各加入终物质的量浓度250μmol/L过氧化氢,空白对照组常规培养。培养24 h后,采用2′,7′-二氯二氢荧光素二乙酸酯荧光探针检测细胞内活性氧水平(以绿色荧光强度表示),细胞计数试剂盒8法检测并计算细胞存活率。(4)取10只6~8周龄雄性BALB/c小鼠(性别、鼠龄下同),分为磷酸盐缓冲液(PBS)组与氧化铜组,每组5只。氧化铜组小鼠经尾静脉注射200 ng/mL的氧化铜纳米酶800 ng/kg,PBS组小鼠注射等体积的PBS。2组小鼠均每天注射1次,连续注射7 d。于第8天,取每组5只小鼠,采血行血细胞与血清生化分析,处死后收集心、肝、脾、肺和肾行苏木精-伊红(HE)染色后组织病理学观察。(5)取20只小鼠分为PBS组与氧化铜组,每组10只。采用链脲佐菌素及高糖高脂饮食法诱导糖尿病,并在其背部制作直径6 mm的全层皮肤缺损创面。伤后即刻,PBS组小鼠创面滴加20μL PBS,氧化铜组小鼠创面滴加20μL 200 ng/mL的氧化铜纳米酶,连续处理12 d。每组选定3�Objective To investigate the effects and mechanism of copper oxide nanozymes on wound healing of full-thickness skin defects in diabetic mice.Methods(1)Copper oxide nanozymes were synthesized through the reaction of copper chloride and L-ascorbic acid.Transmission electron microscope was used for observing the particle size and morphology of copper oxide nanozymes,and dynamic light scattering particle size analyzers and Zeta potentiometer were used to analyze the hydrated particle size and surface potential of copper oxide nanozymes,respectively.(2)The hydrogen peroxide detection kit,superoxide anion determination kit,and 3,3′,5,5′-tetramethylbenzidine were used to test the hydrogen peroxide,superoxide anion,and hydroxyl radicals scavenging ability of 150 ng/mL copper oxide nanozymes,respectively,and the scavenging proportions of hydrogen peroxide,superoxide anion,and hydroxyl radicals were calculated.The sample numbers were all 3.(3)Mouse fibroblast cell line 3T3 cells were divided into blank control group,simple hydrogen peroxide group,and hydrogen peroxide+copper oxide group according to the random number table(the same grouping method below),with 3 wells in each group.Cells in hydrogen peroxide+copper oxide group were pre-treated with copper oxide nanozymes in final mass concentration of 25 ng/mL for 30 minutes,and then hydrogen peroxide in final molarity of 250μmol/L was added into simple hydrogen peroxide group and hydrogen peroxide+copper oxide group.Cells in blank control group were routinely cultured.After 24 hours of culture,2′,7′-dichlorodihydrofluorescein diacetate fluorescence probe was used to detect the level of reactive oxygen species(indicated by green fluorescence intensity)in cells and cell counting kit-8 assay was performed to detect and calculate the cell survival rate.(4)Ten male BALB/c mice aged 6-8 weeks(the same gender and age below)were divided into phosphate buffer saline(PBS)group and copper oxide group,with 5 mice in each group.The mice in the copper oxide group were injected

关 键 词：糖尿病 伤口愈合 氧化性应激 氧化铜纳米酶 

分 类 号：R644[医药卫生—外科学] R63[医药卫生—临床医学]