δ型阿片受体对离体培养人表皮干细胞生物学活性的影响  

Effect of δ-opioid receptor on bioactivity of human epidermal stem cells in vitro

作　　者：程飚[1] 项晓飞[2] 邹吉平[3] 朱江婷[2] 万雨[2] 

机构地区：[1]广州军区广州总医院整形外科、全军热区损伤救治与组织修复重点实验室,510010 [2]南方医科大学研究生学院 [3]第三军医大学研究生学院

出　　处：《中华创伤杂志》2014年第8期816-821,共6页Chinese Journal of Trauma

基　　金：国家自然科学基金资助项目（30871028,81171812）

摘　　要：目的 探讨δ型阿片受体活化对离体人表皮干细胞（human epidermal stem cells,hESCs）增殖与迁移的影响,为临床皮肤损伤的诊治提供理论依据. 方法 取健康青年自愿捐赠的新鲜包皮组织,采用酶消化和差速贴壁法分离培养hESCs,行整合素β1、细胞角蛋白19（cytokeratin 19,CK19）染色,流式细胞仪检测hESCs纯度.取第2代hESCs分别用含1 nmol/L（D-Ala2、K-Leu5）-脑啡肽的角质形成细胞无血清培养基（keratinocyte serum-free medium,K-SFM）（A组）、含1 nmol/L纳曲吲哚和1 nmol/L（D-Ala2、K-Leu5）-脑啡肽的K-SFM培养基（B组）以及单纯K-SFM培养基（C组）连续培养5d.采用MTT法检测hESCs分裂增殖活性.于培养后24 h刮擦生长融合成片的hESCs,制备宽度为100 μm的体外单层hESCs缺损模型,于模型制备后24,48,72,96 h在倒置相差显微镜下观察各组表皮细胞划痕创伤模型愈合情况,计算模型制备后72 h各组创面愈合率. 结果 原代培养的hESCs贴壁后呈鹅卵石样生长.免疫荧光染色法检测第2代hESCs的整合素β1、CK19均呈阳性标记,流式细胞仪检测hESCs的纯度为95.6％.分组培养后,MTT结果显示,A组hESCs分裂增殖活性高于C组,B组低于C组,各组间差异均有统计学意义（P＜0.05）.单层细胞缺损模型制备后24 h,各组均可见hESCs迁移越过创缘,其中A组越过创缘的细胞数多于C组,B组少于C组.模型制备后72 h,细胞迁移A组为（89.5±0.7）％,B组为（76.1±0.3）％,C组为（81.1±0.6）％,组间差异均有统计学意义（P＜0.05）. 结论 δ型阿片受体活化后促进hESCs的分裂增殖和迁移活动,并可能参与皮肤损伤与修复过程.Objective To observe the effect of δ-opioid receptor on proliferation and migration of human epidermal stem cells （hESCs） in vitro so as to offer treatment theory for skin injury.Methods hESCs from fresh foreskin tissues of normal young volunteers were isolated and cultured by enzyme digestion and differential adherence technique.Immunofluorescent staining was used to determine expression of integrin β1 and cytokeratin 19 （CK19） and flow cytometry was used for cell count.Second generation of cells were cultured for 5 consecutive days with keratinocyte serum-free medium （K-SFM） supplemented with 1 nmol/L （D-Ala2,K-Leu5）-enkephalin in Group A,with K-SFM supplemented with 1 nmol/L naltrindole and 1 nmol/L （D-Ala2,K-Leu5）-enkephalin in Group B,and with isolated K-SFM in Group C.Cellular division and proliferation were detected by MTT method.An in vitro 100 μm scratch-wound model was created on the confluent monolayer cells at 24 hours of incubation.Cells migrating from the wound margin were determined by inverted phase contrast microscope at 24,48,72,and 96 hours after wound formation,while wound closure rate was calculated at 72 hours.Results Primary cultured hESCs presented cobblestone-like shape after adherence growth,Immunofluorescence staining showed positive results for integrin β1 and CK19 and cell purity reached 95.6%.Moreover,MTT findings revealed proliferation of hESCs enhanced significantly in Group A,but lowered in Group B as compared to Group C （P 〈 0.05）.hESCs migrated from the wound margin in all groups at 24 hours.However,more migrated cells were seen in Group A than in Group C and less in Group B than in Group C.Rate of wound closure was （89.5 ±0.7）% in Group A,（76.1 ±0.3）% in Group B,and （81.1 ±0.6）% in Group C at 72 hours,indicating significant differences among groups （P 〈 0.05）.Conclusion Activation of δ-opioid receptor promotes the proliferation and migration of hESCs in vitro and may be implicated in wound healing.

关 键 词：表皮 干细胞 受体 阿片样 细胞增殖 

分 类 号：R96[医药卫生—药理学]