miRNA-203转染诱导人表皮干细胞向汗腺细胞分化的研究  

micro RNA-203 INDUCING DIFFERENTIATION OF HUMAN EPIDERMAL STEM CELLS INTO SWEAT GLAND CELLS IN VITRO

作　　者：宋志芳[1] 刘德伍[2] 彭燕[2] 李津[2] 章志伟[2] 宁璞[2] 刘移峰 

机构地区：[1]南昌大学第一附属医院心血管科,南昌330006 [2]南昌大学第一附属医院烧伤中心,南昌330006

出　　处：《中国修复重建外科杂志》2015年第3期343-350,共8页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30560058;81460293);江西省科技厅自主设计组装重点项目(20133BBG70026)~~

摘　　要：目的探讨mi RNA-203转染诱导人表皮干细胞向汗腺细胞分化的可能性及机制。方法取包皮环切术获得的人正常包皮组织5例,采用0.25%胰蛋白酶-EDTA联合消化法分离表皮和真皮,应用Ⅳ型胶原差速贴壁法纯化人表皮干细胞,倒置相差显微镜下观察细胞生长状况,行β1整合素(integrinβ1,ITGB1)、角蛋白19(cytokeratin 19,CK19)、CK1、CK10、CK18、癌胚抗原(carcinoembryonic antigen,CEA)免疫细胞化学染色鉴定。通过脂质体转染将mi RNA-203模拟物导入人表皮干细胞(实验组),转染对照mi RNA模拟物作为对照组。免疫细胞化学染色法对转染后细胞行ITGB1、CK19、CK1、CK10、CK18、CEA单克隆抗体检测鉴定;实时荧光定量RT-PCR检测转染前及转染后72 h的细胞中mi RNA-203、P63、ITGB1、CK19、CK1、CK10、CK18、CEA m RNA相对表达量;Western blot法检测P63、ITGB1、CK19、CK1、CK10、CK18、CEA蛋白相对表达量;并对mi RNA-203的m RNA表达与P63的m RNA和蛋白表达分别行Pearson相关分析。结果转染前人表皮干细胞CK19、ITGB1阳性表达,转染后CK1、CK10、CK18、CEA阳性表达。实验组转染后细胞mi RNA-203 m RNA相对表达量显著高于转染前(P<0.05)。实验组转染后细胞CK1、CK10、CK18、CEA m RNA及蛋白的相对表达量均高于转染前及对照组,而P63、CK19、ITGB1 m RNA及蛋白的相对表达量均低于转染前和对照组,比较差异均有统计学意义(P<0.05)。上述指标对照组与转染前比较差异均无统计学意义(P>0.05)。转染前后mi RNA-203的m RNA表达量与P63的m RNA和蛋白相对表达量均成负相关,转染前相关系数分别为—0.91(t=3.862,P=0.042)及—0.96(t=5.971,P=0.009);转染后相关系数分别为—0.92(t=4.283,P=0.031)及—0.95(t=5.842,P=0.011)。结论 mi RNA-203能诱导人表皮干细胞分化为汗腺细胞,其作用可能是通过靶向抑制P63来实现的。Objective To observe the possibility and mechanism of microRNA （miRNA）-203 inducing the human epidermal stem cells to differentiate into sweat gland cells. Methods Five normal human foreskin tissues were harvested to prepare a single cell suspension by 0.25% trypsin-EDTA digestion method, then the human epidermal stem cells were isolated and cultured by type IV collagen differential adherent method. The cell morphology was observed by inverted phase contrast microscope. The monoclonal antibodies of integrin [31 （ITGB1）, cytokeratin19 （CK19）, CK1, CK10, CK18, and carcinoembryonic antigen （CEA） were used for identification by immunocytochemical staining. Double stranded mimics of has-miR-203 were transfected into the human epidermal stem cells with Lipofectamine 2000 （experimental group） and the human epidermal stem cells transfected with nonsense miRNA mimics served as control group. The monoclonal antibodies of ITGB1, CK19, CK1, CK10, CK18, and CEA were used for identifying the cells after transfection by immunocytochemical staining; the mRNA relative expressions of miRNA-203, P63, ITGB1, CK19, CK1,CK10, CK18, and CEA were detected by real-time fluorescence quantitative RT-PCR before transfected and at 72 hours after transfected. The protein relative expressions of P63, ITGB1, CK19, CK1, CK10, CK18, and CEA were detected by Western blot. The mRNA expression of miRNA-203 and the mRNA and protein expressions of P63 were analyzed respectively with Pearson correlation. Results The CK19 and ITGB1 were positively expressed before transfection, but CK1, CK10, CK18, and CEA were expressed positively after transfection. The mRNA relative expression of miRNA-203 after transfection in experimental group was significantly higher than that before transfection （P〈0.05）. The mRNA and protein relative expressions of CK1, CK10, CK18, and CEA after transfection in experimental group were significantly higher than those before transfection and control group （P〈0.05）, while the mRNA and protein relati

关 键 词：表皮干细胞 汗腺细胞 分化 miRNA-203 P63 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]