Objective: To isolate and culture adipose-derived stem cells (ADSCs), and to study the effects of the conditioned medium of ADSCs (ADSC-CM) treated with insulin on HaCaT cells.
Methods: ADSCs were isolated from adipose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5 x 10(4) cells/mL. The cells were divided into 2 groups: group A in which the cells were incubated in 1 x 10(-7) mol/L insulin for 3 days, and group B in which the cells were not treated with insulin. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor (HGF). HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups: group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1 x 10(-7) mol/L insulin; group D1, 1 mL 2% FBS. Proliferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration ability was measured by in vitro wound-healing assay 0, 12, 24, 36 and 48 hours after culture.
Results: The level of VEGF in groups A and B was (643.28 +/- 63.57) and (286.52 +/- 46.68) pg/mL, respectively, and the level of HGF in groups A and B was (929.95 +/- 67.52) and (576.61 +/- 84.29) pg/mL, respectively, suggesting differences were significant between two groups (P < 0.05). Cell proliferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881 +/- 0.039, 0.804 +/- 0.041, 0.663 +/- 0.027 and 0.652 +/- 0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 (P < 0.01), group A1 was significantly higher than group B1 (P < 0.05). The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23% +/- 1.98%, 8.82% +/- 2.59%, 31.70% +/- 8.85% and 29.60% +/- 8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 (P < 0.05), group B1 was significantly higher than group A1 (P < 0.05). The migration distance of HaCaT cells in groups A1, B1, C1 and D1 at 36 hours was (0.184 6 +/- 0.019 2), (0.159 8 +/- 0.029 4), (0.059 2 +/- 0.017 6) and (0.058 2 +/- 0.012 3) mm, respectively, whereas at 48 hours, it was (0.231 8 +/- 0.1740), (0.205 1 +/- 0.012 1), (0.079 2 +/- 0.008 1) and (0.078 4 +/- 0.011 7) mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours (P < 0.01), group A1 was significantly higher than group B1 (P < 0.05) at 36 and 48 hours, no significant difference was evident at other time points (P > 0.05).
Conclusion: ADSCs treated with insulin can significantly promote the proliferation and the migration of HaCaT cells and inhibit their apoptosis.