OBJECTIVETo explore the expression of integrin-linked kinase (ILK) in fibroblasts (Fbs) of scar induced by cobalt chloride (CoCl2) and its effect on cell proliferation.

METHODSThe human hypertrophic scar Fbs of seven patients were isolated and cultured in vitro. Cells from the 5th to the 6th passages were used in the experiment. Six bottles of Fbs were obtained from each of the seven patients, and they were respectively cultured with DMEM nutrient solution containing CoCl2 in the concentration of 0, 50, 100, 150, 200, and 250 µmol/L for 24 h. The expression of ILK mRNA was determined with real-time fluorescence quantitative PCR. Fbs were stimulated by CoCl2 in the most suitable concentration (100 µmol/L) and the protein expression of ILK was determined 0, 1, 2, 4, 12, and 24 h after the stimulation. Then the Fbs were divided into control group (cultured with nutrient solution), negative control group (transfected with con-siRNA), and ILK siRNA group (transfected with ILK siRNA). They were cultured with nutrient solution containing CoCl2 in different concentrations 24 h after transfection, with 4 wells for each concentration in each group. The cell proliferation was detected by XTT assay. Data were processed with one-way analysis of variance (ANOVA) and ANOVA for repeated measurement, and LSD method was used in multiple comparisons.

RESULTSThe expression level of ILK mRNA was highest in Fbs cultured with 100 µmol/L CoCl2 for 24 h, with significant difference compared with those of Fbs cultured with other concentrations of CoCl2 (F = 50.958, P < 0.001). The expression of ILK protein in Fbs cultured with 100 µmol/L CoCl2 for 1 h (0.243 ± 0.009) was lower than that cultured for 0 h (0.387 ± 0.017), and it started to increase from 2 h (0.361 ± 0.010), and exaggerated at 4 h (0.584 ± 0.028), 12 h (0.730 ± 0.029), and 24 h (0.785 ± 0.031). The expression levels of ILK protein at 1, 4, 12, 24 h were statistically different from that at 0 h (P values all below 0.05). XTT showed that cell proliferation level was highest in control group when cultured with 100 µmol/L CoCl2 (F = 488.026, P < 0.001), which decreased from 150 µmol/L. The cell proliferation level in control group cultured with 250 µmol/L CoCl2 was significantly lower than that with 0 µmol/L (P values all below 0.05). There was no significant change in cell proliferation in ILK siRNA group among different concentrations of CoCl2 (F = 2.542, P = 0.056). The cell proliferation level in ILK siRNA group was significantly lower than that in control group and negative control group (F = 2519.542, P < 0.001).

CONCLUSIONSILK may be a key protein in response of hypoxia in Fbs. The mild hypoxia can stimulate the expression of ILK and promote the proliferation of Fbs, while severe hypoxia can reduce the expression of ILK and inhibit cell proliferation.