复合人表皮干细胞的猪脱细胞真皮基质对裸鼠全层皮肤缺损创面愈合的影响  

Effects of porcine acellular dermal matrix combined with human epidermal stem cells on wound healing of full-thickness skin defect in nude mice

作　　者：赵小红 郭熠城 陈泓豪 李雪 王颖 倪文强 邢孟秋 张锐 余时沧 潘银根[4] 詹日兴 罗高兴 Zhao Xiaohong;Guo Yicheng;Chen Honghao;Li Xue;Wang Ying;Ni Wenqiang;Xing Mengqiu;Zhang Rui;Yu Shicang;Pan Yingen;Zhan Rixing;Luo Gaoxing(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 400038,China;Department of Stem Cell and Regenerative Medicine,the First Affiliated Hospital of Army Medical University(the Third Military Medical University),Chongqing 400038,China;Department of Mechanical Engineering,University of Manitoba,Winnipeg R3T2N2,Canada;Department of Burns and Plastic Surgery,Qidong People's Hospital,Qidong 226200,China)

机构地区：[1]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400038 [2]陆军军医大学(第三军医大学)第一附属医院干细胞与再生医学科,重庆400038 [3]加拿大曼尼托巴大学机械工程系,温尼伯R3T2N2 [4]江苏省启东市人民医院烧伤整形外科,启东226200

出　　处：《中华烧伤与创面修复杂志》2022年第1期45-56,共12页Chinese Journal of Burns And Wounds

基　　金：重庆市2020年科卫联合医学科研项目(2020MSXM001);陆军军医大学军事医学高新技术培育项目(CX2019JS206)。

摘　　要：目的探讨复合人表皮干细胞(ESC)的猪脱细胞真皮基质(ADM)对裸鼠全层皮肤缺损创面愈合的影响。方法采用实验研究方法。采用数码相机拍照观察猪ADM形态,采用苏木精-伊红(HE)染色观测猪ADM中细胞残留,采用扫描电子显微镜观察猪ADM表面结构,采用红外光谱仪分析猪ADM二级结构,采用动态光散射粒度分析仪分析猪ADM粒径,采用纳米粒度电位仪分析猪ADM电位。采用倒置荧光显微镜观察猪ADM在培养基中放置30 min、1 d、5 d的形态(样本数为2);采用随机数字表法(分组方法下同)将猪ADM分为5 min组、10 min组、20 min组、30 min组、60 min组、120 min组,常温静置相应时间,称量法计算吸水量(样本数为3);将Swiss小鼠胚胎成纤维细胞(Fb)分为仅有培养基的空白对照组和另加入相应终质量浓度的ADM浸提液的50.0 g/L ADM浸提液组、37.5 g/L ADM浸提液组、25.0 g/L ADM浸提液组、12.5 g/L ADM浸提液组、6.5 g/L ADM浸提液组,分别于培养24、48、72 h,采用细胞计数试剂盒8法检测细胞增殖率并进行细胞毒性分级(样本数为5);将1只6周龄雄性SD大鼠红细胞分为生理盐水组、超纯水组及添加相应终质量浓度猪ADM浸提液的5 mg/mL ADM浸提液组、10 mg/mL ADM浸提液组、15 mg/mL ADM浸提液组,于反应3 h,采用酶标仪检测血红蛋白的吸光度值代表猪ADM血液相容性(样本数为3)。从陆军军医大学(第三军医大学)第一附属医院泌尿外科2020年7月收治的1名6岁健康男童包皮环切术后废弃包皮中分离培养ESC并采用流式细胞术鉴定。混合培养法构建复合ESC的猪ADM(以下简称ESC/ADM)颗粒,培养3 d后采用HE染色和激光扫描共聚焦显微镜观察ESC/ADM复合效果。将36只7~8周龄雄性无胸腺裸鼠分成单纯磷酸盐缓冲液(PBS)组、单纯ADM组、单纯ESC组、ESC/ADM组,每组9只,并建立全层皮肤缺损创面模型,伤后即刻,创面均一次性采用相应试剂处理。分别于伤后1、7、1Objective To explore the effects of porcine acellular dermal matrix(ADM)combined with human epidermal stem cells(ESCs)on wound healing of full-thickness skin defect in nude mice.Methods The morphology of porcine ADM was analyzed by photograph of digital camera,the cell residues in porcine ADM were observed by hematoxylin-eosin(HE)staining,the surface structure of porcine ADM was observed by scanning electron microscope,the secondary structure of porcine ADM was analyzed by infrared spectrometer,the porcine ADM particle size was analyzed by dynamic light scattering particle size analyzer,and the porcine ADM potential was analyzed by nano-particle size potentiometer.The morphology of porcine ADM was observed by inverted fluorescence microscope when it was placed in culture medium for 30 min,1 d,and 5 d(n=2).The porcine ADM was divided into 5 min group,10 min group,20 min group,30 min group,60 min group,and 120 min group according to the random number table(the same grouping method below)in static state at normal temperature for the corresponding time to calculate the water absorption by weighing method(n=3).Swiss white mouse embryonic fibroblasts(Fbs)were divided into blank control group(culture medium only),and 50.0 g/L ADM extract group,37.5 g/L ADM extract group,25.0 g/L ADM extract group,12.5 g/L ADM extract group,and 6.5 g/L ADM extract group which were added with the corresponding final concentrations of ADM extract respectively.At post culture hour(PCH)24,48,and 72,the cell survival rate was detected by cell counting kit 8 and the cytotoxicity was graded(n=5).The erythrocytes of a 6-week-old male Sprague-Dawley male rat were divided into normal saline group,ultra-pure water group,and 5 mg/mL ADM extract group,10 mg/mL ADM extract group,and 15 mg/mL ADM extract group which were treated with the corresponding final concentrations of porcine ADM extract respectively.After reaction for 3 h,the absorbance value of hemoglobin was detected by microplate reader to represent the blood compatibility of porcine ADM(n=3

关 键 词：伤口愈合 干细胞研究 皮肤 表皮干细胞 复合基质 

分 类 号：R644[医药卫生—外科学]