异体小鼠脂肪源性间充质干细胞-微孔化羊脱细胞真皮基质对小鼠全层皮肤缺损创面愈合的影响及相关机制  

Effects of allogeneic mouse adipose-derived mesenchymal stem cell-microporous sheep acellular dermal matrix on healing of wound with full-thickness skin defect in mouse and the related mechanism

作　　者：曹胜军[1] 王凌峰[1] 巴特[1] 符雪[1] 李芳 郝春光[1] Cao Shengjun;Wang Lingfeng;Ba Te;Fu Xue;Li Fang;Hao Chunguang(Department of Burns,Burn Research Institute of Inner Mongolia,the Third Affiliated Hospital of Inner Mongolia Medical University,BaoTou 014010,China)

机构地区：[1]内蒙古医科大学第三附属医院烧伤科、内蒙古烧伤研究所,包头014010

出　　处：《中华烧伤杂志》2018年第12期901-906,共6页Chinese Journal of Burns

基　　金：内蒙古自治区自然科学基金(2016MS0811);内蒙古医科大学科技百万工程项目(YKD2015KJBW014)。

摘　　要：目的探讨异体小鼠脂肪源性间充质干细胞(ADSC)-微孔化羊脱细胞真皮基质(ADM)对小鼠全层皮肤缺损创面愈合的影响及相关机制。方法取1只昆明小鼠,断颈处死后取腹股沟处脂肪组织,体外分离培养小鼠ADSC,取第3代细胞行细胞成脂、成骨诱导分化鉴定,采用流式细胞仪检测CD34、CD73、CD90、CD105的表达。取1只绵羊,宰杀后取背部皮肤,脱细胞处理和冻融法制备微孔化羊ADM。取36只昆明小鼠,均在背部制备1个直径为12mm的圆形全层皮肤缺损创面,以微孔化羊ADM覆盖创面。术后将小鼠按随机数字表法分为ADSC组和对照组,每组18只。将0.2mL的悬浮有1×10^个ADSC的DMEM/F12培养液注人ADSC组小鼠微孔化羊ADM与创面之间,将0.2mL的DMEM/F12培养液注入对照组小鼠微孔化羊ADM与创面之间。术后12、17d,计算2组小鼠创面愈合率;术后7、12、17d,背光反向照射下观察2组小鼠创面血管生长情况;术后7、12、17d,行苏木素-伊红染色观察ADSC组小鼠创面肉芽组织,并于术后7d测量2组小鼠创面肉芽组织厚度;术后12、17d,采用免疫组织化学法检测2组小鼠创面血管内皮生长因子(VEGF)表达。以上实验中各组各时间点样本数均为6。对数据行t检验、析因设计方差分析。结果(1)成脂诱导7d,经油红O染色细胞质可见红色小油滴;成骨诱导21d,硝酸银染色培养基内可见黑色的钙盐沉积。细胞CD73、CD90、CD105、CD34表达率分别为97.82%、99.32%、97.35%、5.88%。细胞鉴定为ADSC。(2)术后12、17d,ADSC组创面愈合率[(78±6)%、(98±3)%]均高于对照组[(60±9)%、(90±4)%,t=4.26、4.46,P<0.01]。(3)术后7d,2组小鼠都无明显的向创面中心生长的血管,但ADSC组的创面肉芽组织已全部覆盖创面;术后12d,ADSC组小鼠创面比对照组血管更加丰富;术后17d,ADSC组小鼠创面可见粗大的血管且贯穿整个创面,对照组也可见粗大的血管生成,但未贯穿创面。(4)ADSC组小鼠术后7d创�Objective To explore the allogeneic mouse adipose-derived mesenchymal stem cell (ADSC)-microporous sheep acellutar dermal matrix (ADM)on healing of wound with full-thickness skin de- feet in mouse and the related mechanism.Methods One Kunming mouse was sacrificed by cervical dislo- cation to collect adipose tissue from inguinal region.Mouse ADSCs were isolated from the adipose tissue and cultured in vitro.Cells of the third passage were identified by cell adipogenie and osteogenic differentiation. The expressions of CD73,CD90,CD105,and CD34were analyzed by flow cytometry.After one sheep was sacrificed,microporous sheep ADM was prepared from sheep back using deeellularization method and freezing-thawing method.A 12mm diameter,round,full-thickness skin defect wound was made on the back of each one of 36Kunming mice.The wounds were covered-by microporous sheep ADM.The mice were divided into group ADSC and control (C)group with 18mice in each group according to the random number table after surgery.A volume of 0.2mL DMEM/FI2culture medium containing 1×10^6ADSCs was injected between mieroporous sheep ADM and wound of mice in group ADSC.While 0.2mL DMEM/F12culture medi- um was injected between microporous sheep ADM and wound of mice in group C.On post surgery day (PSD)12and 17,wound healing rates of mice in the 2groups were calculated.On PSD 7,12,and 17, wound vascularization of mice in the 2groups was observed under reverse irradiation of baeklight.On PSD 7, 12,and 17,the wound granulation tissue of mice in group ADSC was observed by hematoxylin and eosin staining.On PSD 7,the thicknesses of granulation tissue of mice in the 2groups was measured.On PSD 12 and 17,expressions of VEGF in wounds of mice in the 2groups were detected by immunohistoehemieal method.The sample number was 6in each group at each time point in the above experiments.Data were processed with t test and analysis of variance of factorial design.Results (1)After 7days of adipogenie induction,lipid droplet was observed in cytoplasm using oil red O sta

关 键 词：创伤和损伤 间质干细胞移植 生物敷料 伤口愈合 脂肪源性间充质干细胞 脱细胞真皮基质 

分 类 号：R641[医药卫生—外科学]