白细胞介素17修饰小鼠骨髓间充质干细胞对异体小鼠皮肤移植排斥反应的影响及机制  

Effects and mechanism of interleukin-17-modified mouse bone marrow mesenchymal stem cells on rejection reaction of allogeneic skin transplantation in mice

作　　者：马腾霄[1] 许颖溦 姜笃银[2] Ma Tengxiao;Xu Yingwei;Jiang Duyin(Department of Plastic Surgery,Henan Provincial People's Hospital,Zhengzhou 450000,China;Emergency Department,the Second Hospital of Shandong University,Jinan 250000,China)

机构地区：[1]河南省人民医院整形外科,郑州450000 [2]山东大学第二医院急诊科,济南250000

出　　处：《中华烧伤杂志》2020年第3期234-243,共10页Chinese Journal of Burns

基　　金：国家自然科学基金(81873934)。

摘　　要：目的探讨白细胞介素17(IL-17)修饰的小鼠骨髓间充质干细胞(BMSC)对异体小鼠皮肤移植的影响及机制。方法(1)取5只BALB/c小鼠(均为雌性,4~8周龄,性别、鼠龄下同),处死后取股骨、胫骨和肱骨,采用全骨髓密度梯度离心联合贴壁分离法分离纯化培养BMSC,取第3代细胞行形态学观察,该代细胞经成骨、成脂诱导分化鉴定。第4代细胞经干细胞表面标志物鉴定。取第3~6代BMSC,经终质量浓度50 ng/mL小鼠重组IL-17预处理5 d后,行形态学观察。取IL-17预处理的BMSC和未经IL-17预处理BMSC标记碳花青荧光染料(CM-Dil),行形态学观察并计算标记率。(2)取45只C57BL/6J小鼠,按随机数字表法分为磷酸盐缓冲液(PBS)对照组13只、单纯BMSC组16只、BMSC+IL-17组16只。在小鼠皮肤移植术前1 d,单纯BMSC组13只小鼠经尾静脉注射5×10^6个/mL未经CM-Dil标记的BMSC 0.1 mL,另3只小鼠经尾静脉注射5×10^6个/mL CM-Dil标记的BMSC 0.1 mL;BMSC+IL-17组13只小鼠经尾静脉注射5×10^6个/mL未经CM-Dil标记的IL-17预处理的BMSC 0.1 mL,另3只小鼠经尾静脉注射5×10^6个/mL CM-Dil标记的IL-17预处理的BMSC 0.1 mL;PBS对照组13只小鼠经尾静脉注射0.1 mL PBS。取45只BALB/c小鼠作供体,将前述3组处理后的45只C57BL/6J小鼠作为受体,建立背-背全厚皮移植模型。于移植术后第2天,再次对3组小鼠注射与移植术前1 d一样的等量相应细胞或PBS。于移植术后第7天,分别取单纯BMSC组和BMSC+IL-17组中注射CM-Dil标记的BMSC和CM-Dil标记的IL-17预处理BMSC的3只小鼠,脱颈处死后采用荧光显微镜观察BMSC的CM-Dil示踪并计数。于移植术后第6天拆除敷料后,从单纯BMSC组注射未经CM-Dil标记的BMSC的13只小鼠和BMSC+IL-17组注射未经CM-Dil标记的IL-17预处理BMSC的13只小鼠及PBS对照组13只小鼠中选取7只小鼠,记录移植皮片的存活时间。于移植术后第8天,从3组剩余的6只小鼠中分别取3只小鼠进行移植皮片的大体观察Objective To explore the effects and mechanism of interleukin-17(IL-17)-modified mouse bone marrow mesenchymal stem cells(BMSCs)on the allogeneic skin transplantation in mice.Methods(1)The femur,tibia,and humerus were isolated from five BALB/c mice(all female,aged 4 to 8 weeks,the same gender and age below)after sacrifice.BMSCs were isolated,purified,and cultured by whole bone marrow density gradient centrifugation combined with adherent separation method.The third passage of cells was used for morphological observation and identification of adipogenic and osteogenic differentiation.The fourth passage of cells was used for identification of the expression of stem cell surface markers.The third to sixth passages of BMSCs were pretreated with mouse recombinant IL-17 at a final mass concentration of 50 ng/mL for 5 days,and then were harvested for morphological observation.After being labeled with carbocyanine fluorescent dye(CM-Dil),IL-17-pretreated BMSCs and IL-17-unpretreated BMSCs were obtained for morphological observation and the labeling rates were calculated.(2)Forty-five C57BL/6J mice were divided into phosphate buffer solution(PBS)control group(n=13),BMSCs alone group(n=16),and BMSCs+IL-17 group(n=16)according to the random number table.One day before the skin transplantation of mice,0.1 mL BMSCs(5×10^6 cells/mL)without CM-Dil labeling were injected to the 13 mice in BMSCs alone group through the tail vein,and 0.1 mL BMSCs(5×10^6 cells/mL)labeled with CM-Dil were injected to the other 3 mice in BMSCs alone group through the tail vein.IL-17-pretreated BMSCs(5×10^6 cells/mL)without CM-Dil labeling in the volume of 0.1 mL were injected to the 13 mice in BMSCs+IL-17 group through the tail vein,and 0.1 mL IL-17-pretreated BMSCs(5×10^6 cells/mL)labeled with CM-Dil were injected to the other 3 mice in BMSCs+IL-17 group through the tail vein.PBS in the volume of 0.1 mL was injected to the 13 mice in PBS control group through the tail vein.Forty-five BALB/c mice were used as donors,and forty-five treated C57BL/6

关 键 词：白细胞介素17 间质干细胞 免疫耐受 皮肤移植 

分 类 号：R34[医药卫生—基础医学] R329.2