异体表皮干细胞对裸鼠全层皮肤缺损创面移植异体全厚皮成活的影响及其机制  

Effects and mechanisms of allogeneic epidermal stem cells on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds

作　　者：黄韶斌 胡志成[1] 张逸[2] 唐冰[1] 王鹏[1] 徐海琳 王志勇 董云先 程璞 荣燕超 吴军 朱家源[1] Huang Shaobin;Hu Zhicheng;Zhang Yi;Tang Bing;Wang Peng;Xu Hailin;Wang Zhiyong;Dong Yunxian;Cheng Pu;Rong Yanchao;Wu Jun;Zhu Jiayuan(Department of Burns,the First Affiliated Hospital of Sun Yat-sen University,Guangzhou 510080,China;Department of Burns and Plastic Surgery,Affiliated Hospital of Nantong University,Nantong 226001,China;Department of Burns and Plastic Surgery,the First Affiliated Hospital of Shenzhen University,Shenzhen 518037,China)

机构地区：[1]中山大学附属第一医院烧伤科,广州510080 [2]南通大学附属医院烧伤整形外科,226001 [3]深圳大学第一附属医院烧伤整形科,518037

出　　处：《中华烧伤杂志》2021年第11期1061-1069,共9页Chinese Journal of Burns

基　　金：国家自然科学基金面上项目(81871565);广东省科技计划(2016B090916001);广东省基础与应用基础研究基金(2019A1515012208);中山大学临床医学研究5010计划(2013001,2018003);中山大学高校基本科研业务费(19ykpy66)。

摘　　要：目的探讨异体表皮干细胞(ESC)对裸鼠全层皮肤缺损创面移植异体全厚皮成活的影响及其机制。方法采用实验研究方法。采用酶消化法从1只4周龄雄性BALB/c-NU裸鼠(品系、鼠龄与性别下同)中获取培养7 d呈铺路石状原代ESC,其第3代细胞经流式细胞仪鉴定阳性表达ESC标志物CD44且阴性表达CD45,经免疫荧光法鉴定阳性表达ESC标志物p63与整合素6α且阴性表达CD71。取对数生长期的第3代ESC进行后续实验。取26只裸鼠,采用随机数字表法平均分为磷酸盐缓冲液(PBS)组和ESC组,在每只裸鼠背部制备全层皮肤缺损创面后,2组创面分别喷涂等体积的PBS、ESC,创面上均移植从另外4只裸鼠背部切取制备的全厚皮。分别取2组10只裸鼠,观察术后0(即刻)、3、7、14、21 d创面愈合与皮片存活情况并计算术后3、7、14、21 d皮片存活比和皮片收缩率(样本数为各时间点存活皮片数),采用激光散斑血流成像仪检测术后3、7、14 d皮片的血流灌注情况并计算各时间点ESC组与PBS组裸鼠皮片血流灌注比(样本数为各时间点2组配对均存活皮片对数);分别取2组剩余3只裸鼠,取术后7 d皮片组织,分别采用实时荧光定量反转录PCR法和蛋白质印迹法检测组织中肿瘤坏死因子α(TNF-α)、白细胞介素8(IL-8)、IL-10、Ⅰ型胶原、Ⅲ型胶原与基质金属蛋白酶9(MMP-9)的mRNA和蛋白表达。对数据行Log-rank检验、重复测量方差分析、单因素方差分析、独立样本t检验与Bonferroni校正。结果以术后0 d情况为参照,2组裸鼠术后3、7、14、21 d创面逐渐愈合,皮片收缩情况逐渐明显,其中PBS组裸鼠创面收缩愈合情况较ESC组明显。术后3 d,ESC组1只裸鼠皮片移植失败,PBS组3只裸鼠皮片移植失败;术后7 d,PBS组又有1只裸鼠皮片移植失败。2组裸鼠术后3、7、14、21 d皮片存活比相近(P>0.05)。术后3、7、14、21 d,ESC组裸鼠皮片收缩率分别为(9.2±0.4)%、(19.7±1.2)%、(Objective To investigate the effects and mechanisms of allogeneic epidermal stem cells(ESCs)on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds.Methods Experimental research methods were applied.Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse(the same strain,age,and sex below).The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45,meanwhile,the positive expression of ESC markers of p63 and integrin 6α,and negative expression of CD71 were identified by immunofluorescence method.The ESCs of third passage in the logarithmic growth phase were used for the following experiments.Twenty-six nude mice were equally divided into phosphate buffered saline(PBS)group and ESCs group according to the random number table.A full-thickness skin defect wound was made on the back of each nude mouse,and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs,respectively.The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice.Each ten nude mice from the two groups were selected,the wound healing and skin survival on post surgery day(PSD)0(immediately),3,7,14,and 21 were observed,and the survival ratio and shrinkage rate of skin grafts on PSD 3,7,14,and 21 were calculated(the number of sample was the number of surviving skin grafts at each time point);the blood perfusion in the skin grafts on PSD 3,7,and 14 was detected by the laser speckle blood flow imager,and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated(the number of sample was the pair number of surviving skin grafts in group pairing at each time point).The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7,and the mRNA expressions and protein expressions of tumor necrosi

关 键 词：干细胞移植 皮肤移植 伤口愈合 表皮干细胞 

分 类 号：R632.1[医药卫生—外科学] R605[医药卫生—临床医学]