微小RNA-203下调诱导人角质形成细胞向表皮干细胞去分化的研究  

Down-Regulated Mirna-203 Induces Dedifferentiation of Human Keratinocytes into Epidermal Stem Cells

作　　者：彭燕 宋志芳[2] 刘德伍[3] 李津[3] 章志伟[3] 宁璞[3] PENG Yan;SONG Zhi-fang;LIU De-wu;LI Jin;ZHANG Zhi-wei;NING Pu(Siping Community Health Service Center of Yangpu District,Shanghai 200092,China;Department of Cardiovascular Medicine,the First Affiliated Hospital of Nanchang University,Nanchang 330006,China;Department of Burns Center,the First Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区：[1]上海市杨浦区四平社区卫生服务中心,200092 [2]南昌大学第一附属医院心血管科,江西省南昌市330006 [3]南昌大学第一附属医院烧伤中心,江西省南昌市330006

出　　处：《中国全科医学》2017年第B12期53-58,共6页Chinese General Practice

基　　金：江西省科技厅自主设计组装重点项目(20133BBG70026);江西省教育厅科技项目(GJJ170046)

摘　　要：目的观察微小RNA-203抑制物(miRNA-203 inhibitor)转染诱导人角质形成细胞向表皮干细胞去分化的可能性及机制。方法(1)取包皮环切术后的人正常包皮组织5例,分离培养人角质形成细胞。倒置显微镜下观察细胞的生长状况,采用免疫细胞化学染色法对人角质形成细胞行角蛋白1(CK1)、CK10、CK19、β1整合素(ITGB1)单克隆抗体检测鉴定。(2)通过脂质体Lipofectamine 2000将miR-203单链抑制物分子转染人角质形成细胞作为实验组,将对照micro RNA抑制物转染人角质形成细胞作为对照组。(3)对转染后的实验组和对照组细胞行CK1、CK10、CK19、ITGB1单克隆抗体检测鉴定;采用实时荧光定量RT-PCR技术检测实验组和对照组转染前后细胞miR-203、P63、CK1、CK10、CK19、ITGB1的mRNA表达量;Western blot法检测实验组和对照组转染前后细胞P63、CK1、CK10、CK19、ITGB1的蛋白表达量。(4)对转染前后miR-203的mRNA表达量与P63的mRNA表达量和蛋白表达量分别行Pearson相关分析。结果(1)转染前人角质形成细胞CK1、CK10表达阳性,转染后CK19、ITGB1表达阳性;(2)实验组转染后细胞miR-203、CK1、CK10 mRNA相对表达量较转染前和对照组显著降低(P<0.05),P63、CK19、ITGB1mRNA相对表达量较转染前和对照组明显升高(P<0.05);而对照组与转染前比较无明显变化(P>0.05)。(3)实验组转染后细胞CK1、CK10蛋白相对表达量显著低于转染前和对照组(P<0.05);CK19、ITGB1蛋白相对表达量显著高于转染前和对照组(P<0.05);而对照组与转染前比较无明显变化(P>0.05)。(4)miR-203的基因表达与P63的基因和蛋白表达均成显著负相关,转染前相关系数r值分别为-0.907(t=3.730,P=0.038)及-0.956(t=5.644,P=0.012);转染后r值分别为-0.924(t=4.185,P=0.028)及-0.931(t=4.418,P=0.023)。结论 miR-203下调能诱导人角质形成细胞去分化为表皮干细胞,靶基因p63表达上调可能是其重要机制之一。Objective To observe the possibility and mechanism of dedifferentiation of human keratinocyte to epidermal stem cells induced by transfection with RNA-203 inhibitor.Methods(1)Human foreskin keratinocytes were isolated and cultured from 5 normal prepuce tissues after circumcision.The growth of the cells was observed under inverted microscope.The monoclonal antibodies against keratin 1(CK1),CK10,CK19,and integrinβ1(ITGB1)were used for identification by immunocytochemical staining.(2)Single stranded inhibitors of hsa-miR-203 were transfected into human keratinocytes with Lipofectamine 2000 as the experimental group,and human keratinocytes transfected with microRNA inhibitor were served as control group.(3)After transfection,the cells in experiment group and control group were detected by CK1,CK10,CK19 and ITGB1 monoclonal antibodies.The mRNA expressions of miR-203,P63,CK1,CK10,CK19 and ITGB1 in experiment group and control group were detected by real-time quantitative RT-PCR technique before and after transfection.The expression of P63,CK1,CK10,CK19 and ITGB1 in experiment group and control group before and after transfection were detected by Western blot.The expression of mRNA was measured.The protein relative expression of P63,CK1,CK10,CK19 and ITGB1 in experiment group and control group before and after transfection were detected by Western blot.(4)The mRNA expression of miR-203 and the mRNA and protein expressions of P63 before and after transfection were analyzed respectively with Pearson correlation.Results(1)The CK1 and CK10 were expressed positively before transfection,while CK19 and ITGB1 were positively expressed after transfection.(2)After transfection,the mRNA relative expressions of miR-203,CK1,CK10 in experiment group were significantly lower than those in control group and before transfection(P<0.05).The mRNA relative expressions of P63,CK19 and ITGB1 in experiment group were significantly higher than those in control group and before transfection(P<0.05).The control group did not change significantl

关 键 词：角质形成细胞 表皮干细胞 去分化 微小RNA-203 P63 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]