Effects of severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts

Objective To simulate the environmental factors during the process of formation and evolution of hypertrophic scar, so as to explore the effects of moderate and severe hypoxia and low concentration of serum protein on the function of human hypertrophic scar fibroblasts (HSFs). Methods Human HSFs were routinely cultured. Cells of the 3rd to the 6th passage were divided into 10.0% oxygen+ 10.0% fetal calf serum (FCS), 5.0% oxygen+ 5.0% FCS, and 0.5% oxygen+ 0.5% FCS groups according to the random number table. After being cultured with DMEM nutrient solution with no FCS for 24 h, the cells were cultured with the corresponding volume fraction of oxygen and FCS. Cell proliferation activity was determined with methyl-thiazole-tetrazolium assay (denoted as actual cell number). Content of total collagen was detected with Sirius red staining method (denoted as absorbance value). Protein expression levels of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), transforming growth factor β₁ (TGF-β₁), B-cell lymphoma 2 (Bcl-2), and P53 were determined with Western blotting (denoted as ratio of gray value). Cell apoptosis rate was detected by in situ end labeling method. The sample numbers of each group in the above experiments were all 3. Data were processed with Kruskal-Wallis test and Dunnett test. Results (1) Compared with 11 000±1 306 in 10.0% oxygen+ 10.0% FCS group, the cell proliferation activity was higher in 5.0% oxygen+ 5.0% FCS group (13 290±1 500, P<0.05), but lower in 0.5% oxygen+ 0.5% FCS group (6 999±765, P<0.05). (2) Compared with 0.039 6±0.004 2 in 10.0% oxygen+ 10.0% FCS group, the content of total collagen of cells was higher in 5.0% oxygen+ 5.0% FCS group (0.051 6±0.005 1, P<0.05), but lower in 0.5% oxygen+ 0.5% FCS group (0.015 6±0.002 4, P<0.05). (3) Compared with those in 10.0% oxygen+ 10.0% FCS group, the protein expression levels of HIF-1α, VEGF, TGF-β₁, and Bcl-2 were increased (with P values below 0.05), with no obvious difference in protein expression level of P53 in 5.0% oxygen+ 5.0% FCS group (P>0.05), whereas the protein expression levels of HIF-1α, VEGF, TGF-β₁, and Bcl-2 were decreased (with P values below 0.05), while the protein expression level of P53 was increased in 0.5% oxygen+ 0.5% FCS group (P<0.05). (4) Compared with (1.2±0.9)% in 10.0% oxygen+ 10.0% FCS group, the cell apoptosis rate in 5.0% oxygen+ 5.0% FCS group showed no significant difference [(2.6±0.9)%, P>0.05], while it was significantly increased in 0.5% oxygen+ 0.5% FCS group [(13.3±4.1)%, P<0.05]. Conclusions Severe hypoxia and low concentration of serum protein can inhibit proliferation activity and production of total collagen of human HSFs and induce their apoptosis.