小鼠炎症创面组织匀浆体外模拟创面炎症微环境的可行性研究  

Research on feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice

作　　者：郝艺 杨沁馨 王旗 徐广超 祁放 邓呈亮 魏在荣 王达利 Hao Yi;Yang Qinxin;Wang Qi;Xu Guangchao;Qi Fang;Deng Chengliang;Wei Zairong;Wang Dali(Department of Burns and Plastic Surgery,the Affiliated Hospital of Zunyi Medical University,Zunyi 563003,China)

机构地区：[1]遵义医科大学附属医院烧伤整形外科,563003

出　　处：《中华烧伤杂志》2020年第11期1024-1034,共11页Chinese Journal of Burns

基　　金：国家自然科学基金面上项目(81871570、82072195);贵州省科技计划(黔科合支撑[2016]2910号、黔科合支撑[2020],4Y148号);贵州省组织损伤修复与再生医学2011协同创新中心(黔教合协同创新字[2015]07号)。

摘　　要：目的探讨小鼠炎症创面组织匀浆体外模拟创面炎症微环境的可行性。方法(1)取10只8周龄C57BL/6雄性小鼠,在背部中线两侧用打孔器各取直径1.0 cm的圆形全层皮肤组织,制作正常皮肤组织匀浆上清液。形成全层皮肤缺损创面48 h后,取距创缘2 mm内创面组织,制作炎症创面组织匀浆上清液。取2种组织匀浆上清液,调整总蛋白质量浓度为1 mg/mL,酶联免疫吸附测定法检测肿瘤坏死因子α(TNF-α)含量,样本数为6。(2)取原代人脐带间充质干细胞(hUCMSC)并培养至第3代,培养48 h后提取正常外泌体。另外取第3代hUCMSC,分别加入总蛋白质量浓度为30、50、100μg/mL正常皮肤组织匀浆上清液和炎症创面组织匀浆上清液,培养48 h后,提取30、50、100μg/mL正常蛋白刺激外泌体和30、50、100μg/mL炎症蛋白刺激外泌体。取正常外泌体、30μg/mL正常蛋白刺激外泌体和30μg/mL炎症蛋白刺激外泌体,透射电子显微镜下观察外泌体形态,纳米颗粒跟踪分析仪检测外泌体粒径,蛋白质印迹法检测CD9和CD63表达。(3)取1 d龄C57BL/6小鼠乳鼠20只,分离培养原代成纤维细胞(Fb)和第3代Fb,倒置相差显微镜下观察细胞形态。取第3代Fb,培养2 h,利用细胞爬片法结合免疫荧光法观察波形蛋白的表达。(4)取第3代Fb,按随机数字表法分为对照组,正常外泌体组,30、50、100μg/mL正常蛋白刺激外泌体组及30、50、100μg/mL炎症蛋白刺激外泌体组,每组4孔。对照组不进行任何处理,其余7组依次加入实验(2)中制备的正常外泌体,30、50、100μg/mL正常蛋白刺激外泌体和30、50、100μg/mL炎症蛋白刺激外泌体,并调整外泌体终质量浓度为10μg/mL。培养48 h,采用细胞计数试剂盒8法检测8组细胞活力。(5)取2个批次第3代Fb,同实验(4)进行分组及处理,每组4孔,并分别调整外泌体终质量浓度为1、10μg/mL,采用细胞划痕试验检测培养6、12、24 h细胞迁移率。(6)取2个批次第3Objective To investigate the feasibility of in vitro inflammatory wound microenvironment simulated by using inflammatory wound tissue homogenate of mice.Methods(1)Ten eight-week-old C57BL/6 male mice were collected and full-thickness skin tissue with diameter of 1.0 cm on both sides of the midline of the back was taken with a perforator to make the normal skin tissue homogenate supernatant.At 48 h after the full-thickness skin defect wound was established,the wound tissue within 2 mm from the wound edge was taken to make inflammatory wound tissue homogenate supernatant.Two kinds of tissue homogenate supernatant were taken to adjust the total protein concentration to 1 mg/mL,and the tumor necrosis factorα(TNF-α)content was detected by enzyme-linked immunosorbent assay.The number of sample was 6.(2)The primary passage of human umbilical cord mesenchymal stem cells(hUCMSCs)were collected and cultured to the 3rd passage with the normal exosomes being extracted from the hUCMSCs after cultured for 48 h.Another batch of hUCMSCs in the 3rd passage was collected and stimulated with inflammatory wound tissue homogenate supernatant of 30,50,and 100μg/mL total protein and normal skin tissue homogenate supernatant of 30,50,and 100μg/mL total protein,respectively.After cultured for 48 h,the exosomes stimulated with normal protein of 30,50,and 100μg/mL and exosomes stimulated with inflammatory protein of 30,50,and 100μg/mL were extracted.Normal exosomes,exosomes stimulated with 30μg/mL normal protein,and exosomes stimulated with 30μg/mL inflammatory protein were collected,the morphology was observed by transmission electron microscope,the particle size was detected by nanoparticle tracking analyzer,and the expressions of CD9 and CD63 were detected by Western blotting.(3)Twenty one-day-old C57BL/6 mice were taken to isolate the primary passage of fibroblasts(Fbs)and the 3rd passage of Fbs,whose morphology was observed under the inverted phase contrast microscope.The Fbs of 3rd passage were collected to observe the expres

关 键 词：炎症 成纤维细胞 外泌体 组织匀浆 微环境 人羊膜间充质干细胞 

分 类 号：R644[医药卫生—外科学] R619+.6[医药卫生—临床医学] Q786[生物学—分子生物学]