[Biologic effect of nitric oxide on human epidermal stem cells in vitro].
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.
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.
Ri-xing
RX
Zhan
Wei
W
Sun
Zhi-hui
ZH
Yao
Yan-yan
YY
Cui
Si-si
SS
Yang
Jiang-lin
JL
Tan
Jun-yi
JY
Zhou
Ying
Y
Wang
Jun-jie
JJ
Yang
Xiao-rong
XR
Zhang
Xiao-hong
XH
Hu
Jun
J
Wu
Gao-xing
GX
Luo
D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects; D006801:Humans; D009569:Nitric Oxide / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects
D009569:Nitric Oxide