中文摘要：

目的 观察微小RNA-203与P63在人表皮干细胞和KC中的表达变化,探讨其在表皮增生分化中的作用及意义. 方法 取2013年3-6月在南昌大学第一附属医院泌尿外科行包皮环切术的5例患者的5个正常包皮组织标本,采用胰蛋白酶消化法分离表皮获得单细胞悬液,应用Ⅳ型胶原快速黏附法将细胞分成快速黏附和未快速黏附的细胞分别培养，倒置相差显微镜下观察细胞分离即刻及培养3d的生物学特性,行CD29、角蛋白19、角蛋白1、角蛋白10免疫细胞化学染色鉴定，实时荧光定量RT-PCR法检测微小RNA-203表达和P63的mRNA表达,蛋白质印迹法检测P63蛋白表达，对数据行t检验、Pearson相关分析，结果 （1）细胞分离即刻,快速黏附的细胞群中细胞小而圆,分布较均匀;培养3d,细胞贴壁牢固并呈克隆性生长，细胞分离即刻,未快速黏附的细胞群中细胞形状、大小不一致,分布不均匀;培养3 d,细胞贴壁不牢固且无克隆性生长，快速黏附的细胞中CD29、角蛋白19呈阳性表达,未快速黏附的细胞中角蛋白1、角蛋白10呈阳性表达.经鉴定,快速黏附的细胞为表皮干细胞,未快速黏附的细胞为KC，（2）表皮干细胞微小RNA-203的相对表达量为0.74±0.20,较KC的3.66 ±0.34明显下调（t=16.582,P＜0.001），表皮干细胞P63的mRNA相对表达量为4.16±0.28,较KC的2.90±0.39明显上调（t=5.850,P=0.001），表皮干细胞P63的蛋白相对表达量为1.42 ±0.05,较KC的0.73 ±0.03明显上调（t=26.460,P＜0.001），（3）微小RNA-203表达量与P63的mRNA和蛋白表达量均呈明显负相关（r值分别为-0.94、-0.98,P值均小于0.05）， 结论 微小RNA-203与P63在人表皮干细胞和KC中的表达有明显差异,可能与细胞增殖和分化特性不同密切相关。

英文摘要：

Objective To observe the changes in expression of microRNA-203 and P63 in human epidermal stem cells and KCs,and to investigate their effects and significance in the epidermal proliferation and differentiation.Methods （1） Five normal foreskin tissue specimens were collected from 5 patients by circumcision in Department of Urinary Surgery of the First Affiliated Hospital of Nanchang University from March to June in 2013.Then single cell suspension was obtained by separating epidermis with trypsin digestion method.The cells were divided into quick adherent cells and non-quick adherent cells by type Ⅳ collagen differential adherent method.The biological characteristics of cells were ohserved by inverted phase contrast microscope immediately after isolation and on post euhure day （PCD） 3.The expression of CD29,keratin 19,keratin 1,and keratin 10 was identified by immunocytochemical staining.The expression of microRNA-203 and mRNA of P63 was determined by real-time fluorescent quantitative RT-PCR.The protein expression of P63 was determined by Western blotting.Data were processed with t test and Pearson correlation analysis.Results （1） Immediately after isolation,quick adherent cells were small,round,and dispersed uniformly.On PCD 3,the cells adhered firmly,and they grew in clones.Immediately after isolation,non-quick adherent cells appeared in different shapes and sizes,and dispersed unevenly.On PCD 3,the cells adhered precariously and did not show clonal growth.Quick adherent cells showed positive expression of CD29 and keratin 19,while non-quick adherent cells showed positive expression of keratin 1 and keratin 10.Quick adherent cells were identified as epidermal stem cells,and non-quick adherent cells were identified as KCs.（2） The expression level of microRNA-203 in epidermal stem cells （0.74 ±0.20） was lower than that in KCs （3.66±0.34,t =16.582,P 〈0.001）.The mRNA expression level of P63 in epidermal stem cells （4.16±0.28） was higher than that in KCs （2.90±0.39,t =5.850,P =0