一氧化氮对人表皮干细胞的体外生物学效应

Zhan Ri-xing, Sun Wei, Yao Zhi-hui, Cui Yan-yan, Yang Si-si, Tan Jiang-lin, Zhou Jun-yi, Wang Ying, Yang Jun-jie, Zhang Xiao-rong, Hu Xiao-hong, Wu Jun, Luo Gao-xing

Institute of Burn Research, Southwest Hospital, State Key Laboratory of Trauma, Burns and Combined Injury, Chongqing Key Laboratory for Diseases Proteomics, the Third Military Medical University, Chongqing 400038, China.

Zhonghua Shao Shang Za Zhi. 2012 Apr;28(2):125-9.

OBJECTIVE

To observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro.

METHODS

ESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin β(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test.

RESULTS

(1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin β(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05).

CONCLUSIONS

Exogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.

目的

观察一氧化氮（NO）对人表皮干细胞（ESC）体外黏附、增殖及迁移的影响。

方法

采用改良的IV型胶原快速贴壁法分离培养ESC。（1）在倒置相差显微镜下观察细胞形态。采用蛋白质免疫印迹法和免疫荧光染色法检测细胞整合素β1和细胞角蛋白19（CK19）的表达水平。（2）划痕处理后，将贴壁的ESC分别用浓度为1、10、100、500 μmol/L的一氧化氮（NO）供体S-亚硝基-N-乙酰青霉胺（SNAP）处理。未用SNAP处理的ESC作为对照。在划痕后12小时和24小时检测ESC的迁移率。采用Transwell实验检测上述浓度SNAP处理的ESC的趋化性，并计数迁移的细胞数。（3）将ESC分别用浓度为10、100、500 μmol/L的SNAP处理1小时。未用SNAP处理的ESC作为对照。采用黏附实验检测ESC的黏附情况，并计算黏附抑制率。在处理后0、12、24、48小时用酶标仪测定ESC的增殖情况（以吸光度值表示）。数据采用单因素方差分析和Dunnett t检验进行处理。

结果

（1）培养第5至9天形成小克隆。培养第10至14天，细胞增殖加快。检测到分离的细胞中表达CK19和整合素β1。这些细胞被鉴定为ESC。（2）与未用SNAP处理的ESC相比[（35.7±0.3）%，（45.7±5.0）%]，在划痕后12小时和24小时，浓度为1至100 μmol/L的SNAP处理的ESC迁移增加。100 μmol/L SNAP处理的ESC迁移率最高[分别为（48.8±2.7）%，（82.1±15.8）%，t值分别为8.34、5.10，P值均<0.01]。100 μmol/L SNAP处理后转移至膜上的ESC数量明显多于未用SNAP处理的ESC（t = 9.24，P = 0.00）。（3）100、500 μmol/L SNAP处理的ESC吸光度值明显高于未用SNAP处理的ESC（t值分别为4.30、4.67，P值均为0.00）。在处理后24、48小时，100、500 μmol/L SNAP处理的ESC增殖明显强于未处理的细胞（t值为2.84至8.17，P值均<0.05）。