RESUMO
Objective • To reveal the effect of denatured collagen type on the endothelial cell proliferation, migration, and angiogenesis-related proteins expression. Methods • Human umbilical vein endothelial cells were cultured on the plates coated with normal collagen (normal collagen group), half concentration normal collagen (half collagen group) or denatured collagen (denatured collagen group). CCK-8 assay was performed to test cell proliferation ability two days later. The effect of collagen on cell migration was measured by scratch test. The expressions of membrane-type 1 matrix metalloproteinase (MT1-MMP) and vascular endothelial growth factor (VEGF) were detected by Western blotting. The expressions of angiopoietin 1 (Ang-1) and Ang-2 were measured by ELISA. Results • CCK-8 results showed that the proliferation activity of denatured collagen group was significantly higher than that of normal collagen group and half collagen group (P<0.05). Cell migratory capacity was increased in denatured collagen group, of which the scratch was almost covered after 16 h. The protein expressions of MT1-MMP and VEGF were significantly higher in denatured collagen group than those in the other two groups (P<0.05), in which the concentration of Ang-2 was also higher than that in normal collagen group (P<0.05). However, no significant difference was found in the concentration of Ang-1 among the three groups. Conclusion • Denatured collagen type can promote proliferation, migration and MT1-MMP, VEGF, and Ang-2 expressions in endothelial cells, suggesting that collagen denaturation may play an active role in the process of angiogenesis during wound healing.
RESUMO
Objective@#To investigate the effects of denatured collagen type Ⅰ on differentiation of human fibroblasts into myofibroblasts.@*Methods@#A small amount of normal skin donated by burn patients undergoing scar surgery was collected. Human fibroblasts were obtained by method of explant culture and then sub-cultured. The fourth passage of cells were used in the following experiments. (1) Fibroblasts were divided into normal collagen group and denatured collagen group according to the random number table, with 10 wells in each group. Fibroblasts in normal collagen group were cultured on normal collagen type Ⅰ coated coverslips. Fibroblasts in denatured collagen group were cultured on denatured type Ⅰ collagen coated coverslips. Expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemical method, and the percentage of PCNA positive cells was calculated. (2) Another batch of fibroblasts were grouped and treated as in (1), with 12 wells in each group. Proliferation activity of cells was determined with methyl-thiazolyl-tetrazolium colorimetry method. (3) Another batch of fibroblasts were grouped and treated as in (1), and the microfilament morphology of cells was observed by rhodamine-phalloidin staining. (4) Another batch of fibroblasts were grouped and treated as in (1). Expression of α smooth muscle actin (α-SMA) of cells was detected by immunohistochemical method, and expression of OB-cadherin of cells was detected by immunofluorescence method. (5) Another batch of fibroblasts were divided into normal collagen, denatured collagen, and common coverslips groups according to the random number table, with 6 wells in each group. Fibroblasts in normal collagen and denatured collagen groups were treated as in (1), while fibroblasts in common coverslips group were cultured on coverslips without collagen coating. Expressions of α-SMA and OB-cadherin of cells were determined with Western blotting. (6) Another batch of fibroblasts were grouped and treated as in (5), and then the mRNA expressions of collagen type Ⅰ, collagen type Ⅲ, and α-SMA of cells were determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with t test, one way analysis of variance, and least-significant difference test.@*Results@#(1) The percentage of PCNA positive cells in denatured collagen group was (83±9)%, significantly higher than (29±9)% in normal collagen group (t=13.53, P<0.01). (2) The proliferation activity of fibroblasts in denatured collagen group was 0.32±0.06, significantly higher than 0.25±0.05 in normal collagen group (t=3.06, P<0.01). (3) The microfilament of fibroblasts in normal collagen group was arranged vertically and in parallel way, paralleling the long axis of cells. The microfilament of fibroblasts in denatured collagen group was denser and thicker. (4) Most fibroblasts in normal collagen group showed long shuttle-like shape typically. Morphology of fibroblasts in denatured collagen group changed, and cells were obviously spreading. Expressions of α-SMA and OB-cadherin of fibroblasts in denatured collagen group were stronger than those in normal collagen group. (5) Expressions of α-SMA of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 1.69±0.41, 0.89±0.27, and 1.46±0.42. Expression of α-SMA of fibroblasts in denatured collagen group was significantly higher than that in normal collagen group (P<0.01). Expressions of OB-cadherin of fibroblasts in denatured collagen, normal collagen, and common coverslips groups were respectively 5.17±0.28, 2.21±0.10, and 4.01±0.56. Expression of OB-cadherin of fibroblasts in denatured group was significantly higher than that in normal collagen group (P<0.01). (6) There was no significant difference in mRNA expression of collagen type Ⅰ of fibroblasts in denatured collagen, normal collagen, and common coverslips groups (F=2.71, P>0.05). The mRNA expressions of collagen type Ⅲ and α-SMA of fibroblasts in normal collagen group were significantly lower than those in denatured collagen group (P<0.01).@*Conclusions@#Denatured collagen type Ⅰ may influence the activity of fibroblasts, thus inducing fibroblasts differentiating into myofibroblasts.