Effect of different culture conditions on biological characteristics of fibroblasts derived from human hypertrophic scar / 中华整形外科杂志

ABSTRACT

Objective@#To investigate the effect of different culture conditions on cell proliferation, apoptosis, collagen synthesis and TGF-β1 expression of fibroblasts derived from human hypertrophic scar(HSF).@*Methods@#Fibroblasts were isolated from human hypertrophic scar and cultured in vitro. Cells of passage 4 to 6 were cultured with 10%O2+ 100% culture medium for 48 h, and then divided into four groups, 10%O2+ 100% culture medium group (control group), 7%O2+ 70% culture medium group (experimental group 1), 4%O2+ 40% culture medium group (experimental group 2) and 1%O2+ 10% culture medium group (experimental group 3) according to the different culture conditions. There are 4 samples in each group. MTT assay, Flow cytometer and the Annexin V-FITC/PI double-staining were performed to detect the cell proliferation, S phase cell ratio and cell apoptosis. Hydroxyproline assay kit was adopted to detect the collagen synthesis of the fibroblasts. Protein expression levels of transforming growth factor β1 (TGF -β1)were determined with Western-blotting. SPSS 13.0 software was used to analyze the data. Analysis of variance ( oneway ANOVA) and SNK test were used to evaluate significant differences among these groups.@*Results@#①Compared with control group, the cell proliferation rate was higher in the experimental group 1(P<0.05), while it significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups (F=163.32, P<0.01). ②The collagen synthesis was much higher in experimental group 1 compared with control group (0.56±0.01 vs 0.42±0.01, P<0.05), and was lower in experimental group 3 (0.48±0.01 , P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ③The S phase cell ratio in the four groups were(19.40±0.37)%, (22.83±0.28)%, (20.94±0.38)% and(14.54±0.21)% respectively. Compared with control group, the S phase cell ratio was significantly higher in experimental group 1 and experimental group 2 (P<0.05), while it was significantly lower in experimental group 3(P<0.05). There was a statistically significant difference among groups (F=357.69, P<0.01). ④ The expression of TGF-β1 increased significantly in experimental group 1 and 2(P<0.05)compared with control group. However, experimental group 3 showed less TGF-β1 expression. There was a statistically significant difference among groups (F=357.69, P<0.01). ⑤The ratio of living cells in the four groups was (96.21±0.29)%, (96.61±0. 24)%, (96.61±0.17)% and(95.36±0.15)% respectively. There was no significant difference between control group and experimental group 1 and 2, while it was significantly decreased in experimental group 3 (P<0.05). There was a statistically significant difference among groups(F=12.38, P<0.01).@*Conclusions@#Mild hypoxia and low concentration of culture medium promote cell proliferation, collagen synthesis, TGF-β1 expression and cell viability of fibroblasts derived from hypertrophic scar. However, severe hypoxia and low concentration of culture medium decrease cell biological characteristics and promote cell apoptosis.