Establishing The Model Of Human Hypertrophic Scar Fibroblasts With Mechanical Stimulation

Objective To establish the hypertrophic scar fibroblasts(HSFB)model with human normal skin fibroblasts(NSFB)induced by mechanical stress.Methods Dermal tissues of normal skin and hypertrophic scar from 3 donors were cultured in vitro.Normal skin fibroblasts(NSFB)and hypertrophic scar fibroblasts(HSFB)were divided into the experimental group and the control group.Experimental groups were loaded with 0.1Hz,10% amplitude sine-wave cyclic stretch for 3,5 and 7 days respectively.Control group were cultured without cyclic stretch.Each experiment was repeated 3 times.At the end of experiments,morphological changes were observed with an inverted microscope;collagen type I and transform growth factor ?1(TGF-?1)in culture supernatant was detected by enzyme linked immunosorbent assay(ELISA);SYBR Green real time quantitative PCR detection system(q PCR)was used to detect the expression level of p130Crk-associated substance(P130Cas),Integrin-?1,TGF-?1,procollagen type I and ?-Smooth muscle actin(?-SMA);Protein Integrin-?1 and P130 Cas were detected by Western-Blot(WB)method.The data were expressed as x ?±s.S-N-Ka test was completed when a significant ANOVA was observed(p < 0.05)to determine groups that were significantly different from each other.Results 1)The morphology of cells: cells of the experimental group proliferated actively with apparent orientation.While in the control group,the cell proliferation was slow and without orientation.2)ELISA results: loaded with 5 days,the collagen type I and TGF-?1 in culture supernatant from experiment group of NSFB and HSFB were with no significant differences(P > 0.05),control groups of HSFB and NSFB,the difference was statistically significant(P < 0.05).Loaded for 3 days and 7 days,differences between each group were statistically significant(P < 0.05);loaded for 3 days,the concentrations of collagen type I and TGF-?1 in NSFB experiment group are lower than that of the HSFB control group;while loaded 7 days,the concentrations of collagen type I and TGF-?1 in NSFB experiment group are higher than that of the HSFB control group.3)SYBR Green q PCR results: loaded for 3 days,compared to NSFB control group,P130 Cas,Integrin-?1,TGF-?1 procollagen type I m RNA expression levels of NSFB experiment group were statistically different,(P < 0.05);?-SMA m RNA expression level showed no difference(P > 0.05).When loaded for 5 days,compared with HSFB control group,expression levels of P130 Cas,Integrin-?1,TGF-?1 procollagen type I and ?-SMA of the NSFB experiment group were no significant differences(P>0.05);while loaded for 7 days,the above indexes showed differences(P<0.05).4)WB results: Loaded for 3 days,the expression levels of P130 Cas and Integrin-?1 from NSFB control and experiment group showed no statistically different(P>0.05).When loaded for 5 days,compared to HSFB control group,the expression levels of protein P130 Cas and Integrin-?1 from NSFB experiment group are with no statistically significant difference(P > 0.05),the difference between HSFB and NSFB control group was statistically significant(P < 0.05).While loaded for 7 days,the above indexes showed differences(P<0.05).Conclusion The HSFB model can be successfully constructed with sine-wave cyclic mechanical stress of 0.1Hz and 10% amplitude for 5 days.