OPN诱导小鼠MSCs向表皮细胞分化促进创面愈合的研究  

Osteopontin induces differentiation of mouse marrow mesenchymal stem cells into epithelial cells and promotes the wound healing in vivo

作　　者：王文平[1] 李薇[2] 陈亮[1] 王珍祥[1] 李世荣[1] 

机构地区：[1]第三军医大学西南医院整形美容外科,重庆400038 [2]蚌埠医学院烧伤整形科,安徽蚌埠233030

出　　处：《第三军医大学学报》2015年第7期594-599,共6页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81071563;81272102);重庆市应用开发项目(2014yykf A0158)~~

摘　　要：目的以骨桥蛋白（osteopontin,OPN）基因敲除小鼠及野生型小鼠为研究对象,探讨骨髓间充质干细胞（mesenchymal stem cells,MSCs）分化为表皮细胞的潜能及骨桥蛋白在分化过程中的作用。方法体外实验：将分离培养的野生型（WT）和基因敲除型（KO）新生鼠（鼠龄1 d）MSCs鉴定纯度后分别设为WT组和KO组,取其第3代分别在表皮转化培养体系中孵育,通过镜下观察、流式细胞术、免疫荧光染色、Western blot等方法,比较两组表皮细胞标志物（CK14）表达水平的差异。动物实验：实验分4组,WT组小鼠创周注射GFP-MSCs（WT/MSCs）组,WT小鼠创周注射PBS（WT/PBS）组,KO小鼠创周注射GFP-MSCs（KO/MSCs）组,KO小鼠创周注射PBS（KO/PBS）组,每组各8只雄鼠,鼠龄6～8周,体质量20～25 g,按分组将分离培养的GFP-MSCs和PBS注射到创周,观察创面愈合及GFP-MSCs向表皮细胞分化的情况。结果分离培养的MSCs呈梭形或成纤维细胞样生长,流式细胞术检测MSCs的纯度超过91%;诱导分化第12、17天的两组细胞检测发现,WT组和KO组的细胞均表达CK14;第17天免疫荧光结果示WT组CK14的表达阳性率（0.936 3±0.009 5）明显高于KO组（0.647 5±0.023 1）,（P〈0.01）;Western blot检测结果也显示KO组的CK14表达量（0.389 4±0.016 8）明显低于WT组（0.614 6±0.015 3）,（P〈0.01）。动物实验WT/PBS组创面愈合速度快于KO/PBS组（P〈0.05）,WT/MSCs组的创面愈合速度快于WT/PBS组（P〈0.05）,注射的GFP-MSCs向表皮细胞分化的能力WT/MSCs组强于KO/MSCs组（P〈0.05）。结论 OPN在体内、体外均可诱导MSCs分化为表皮细胞,进而促进创面愈合。Objective To determine the role of osteopontin （OPN） in the differentiation of mouse marrow mesenchymal stem cells （MSCs） into epithelial cells （OPN） with knockout mice and wild type mice as experiment objects. Methods In vitro experiment： the MSCs were isolated and cultivated from wild type （WT） and knockout （KO） newborn mice （aged 1 day old） and identified for the purity. The cells at the third-generation were cultured to join epithelial transformation culture system. Microscopy, flow cytometry, immunofluorescence staining and Western blotting were used to observe and compare the expression levels of epidermal cells markers （CK14） of the 2 groups. Animal experiment： the experiment was divided into 4 groups, such as WT mice receiving GFP-MSCs injection around the wound （WT/MSCs group）, WT mice injected with PBS （WT/PBS group）, KO mice injected with GFP-MSCs （KO/MSCs group）, and KO mice injected with PBS （ KO/PBS group）. Each group had 8 male mice, at the age of 6 to 8 weeks and weighing 20 to 25 g. According to the grouping, cultivated GFP-MSCs and PBS were injected around the wound, and the wound healing and differentiation of GFP-MSCs into epithelial cells were observed. Results The isolated and cultured MSCs were seen in fusiform- or fibroblast-like. Flow cytometry confirmed the purity of separated MSCs was over 91%. CK14 was expressed in both WT and KO cells after induction for 12 and 17 d. Immunofluorescence staining also showed that the WT group had obviously higher expression level of CK14 than the KO group on the 17th day （0. 936 3± 0. 009 5 vs 0. 647 5± 0. 023 1, P 〈 0.01）, and so was Western blot analysis （0.614 6±0.015 3 vs 0.389 4 ±0.016 8, P 〈0.01）. Animal experiments also showed wound healing of WT/PBS group was significantly faster than the KO/PBS group （ P 〈 0.05 ）. Moreover, the wound healing of WT/MSCs group was faster than WT/PBS group （P 〈 0.05 ） and the capacity of injected GFP- MSCs to differentiate into epithelial

关 键 词：愈合 间充质干细胞(MSCs) 骨桥蛋白(OPN) 表皮 分化 

分 类 号：R322.991[医药卫生—人体解剖和组织胚胎学] R392.21[医药卫生—基础医学]