[微小RNA-203和P63在人表皮干细胞和角质形成细胞中的表达]

Song Zhi-fang, Liu Dewu, Peng Yan, Li Jin, Zhang Zhiwei, Ning Pu, Hu Yanghong

Zhonghua Shao Shang Za Zhi. 2014 Aug;30(4):344-8.

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 observed by inverted phase contrast microscope immediately after isolation and on post culture 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 =0.001). The protein expression level of P63 in epidermal stem cells (1.42 ± 0.05) was higher than that in KCs (0.73 ± 0.03, t =26.460, P <0. 001). (3) The expression level of microRNA-203 was in significantly negative correlation with the expression levels of mRNA and protein of P63 (with r values respectively - 0. 94 and -0.98 , P values below 0.05).

CONCLUSIONS

The expression levels of microRNA-203 and P63 in human epidermal stem cells and KCs were significantly different, which might be related to the different characteristics of proliferation and differentiation of the cells.

目的

观察微小RNA-203和P63在人表皮干细胞和角质形成细胞中的表达变化，探讨其在表皮增殖和分化中的作用及意义。

方法

（1）2013年3-6月，收集南昌大学第一附属医院泌尿外科5例患者因包皮环切术取下的5份正常包皮组织标本。采用胰蛋白酶消化法分离表皮获得单细胞悬液。通过IV型胶原差异贴壁法将细胞分为快速贴壁细胞和非快速贴壁细胞。分离后及培养后第3天，立即用倒置相差显微镜观察细胞生物学特性。采用免疫细胞化学染色鉴定CD29、角蛋白19、角蛋白1和角蛋白10的表达。采用实时荧光定量RT-PCR检测微小RNA-203的表达及P63的mRNA。采用蛋白质印迹法检测P63的蛋白表达。数据采用t检验和Pearson相关分析进行处理。

结果

（1）分离后立即观察，快速贴壁细胞小、圆形，均匀分散。培养后第3天，细胞贴壁牢固，呈克隆生长。分离后立即观察，非快速贴壁细胞形态和大小各异，分散不均匀。培养后第3天，细胞贴壁不牢固，未显示克隆生长。快速贴壁细胞CD29和角蛋白19表达阳性，而非快速贴壁细胞角蛋白1和角蛋白10表达阳性。快速贴壁细胞被鉴定为表皮干细胞，非快速贴壁细胞被鉴定为角质形成细胞。（2）表皮干细胞中微小RNA-203的表达水平（0.74±0.20）低于角质形成细胞（3.66±0.34，t=16.582，P<0.001）。表皮干细胞中P63的mRNA表达水平（4.16±0.28）高于角质形成细胞（2.90±0.39，t=5.850，P=0.001）。表皮干细胞中P�3的蛋白表达水平（1.42±0.05）高于角质形成细胞（0.73±0.03，t=26.460，P<0.001）。（3）微小RNA-203的表达水平与P63的mRNA和蛋白表达水平呈显著负相关（r值分别为-0.94和-0.98，P值均<0.05）。