| Objective:To investigate USP4 in the important role of hyperplastic scar formation,especially in the regulation of TGF-β/Smad signaling pathway, to ensure the correlation of USP4, TβRI and Smad7 and elucidate the mechanism of hypertrophic scar by inhibiting USP4. It will provide important theoretical basis to cure hyperplastic scar as a drug target for USP4.Methods:1. Collecting 15 specimens of HS and normal skin tissue in HS patients from our plastic surgery puts in low temperature, which is quickly taken back to the lab(six months after injury). A part of tissue is washed by the saline with 4% neutral formaldehyde fixed and is dehydrated conventionally, embedded in paraffin and immunohistochemical staining; Western blot for detecting the different expression level of USP4 and TβRI in HS tissue and normal skin tissue.2. Hypertrophic scar fibroblasts(HSFB) and normal skin fibroblasts(NSFB)were cultured in vitro by using tissue combined with modified trypsin digestion with HS tissue and normal skin tissue. The third to the fifth generation of HSFB and NSFB in logarithmic growth phase were selected in the experiment.(1)take the fourth generation of FB, Western blot to detect USP4 and TβRI and Smad7 protein expression level of hyperplastic scar fibroblasts and normal skin fibroblasts;(2)Hyperplastic scar fibroblasts were cultured in cell culture plate for 24 hours. After that, HSFB were intervened by 5μmol/L Vialinin A which was the inhibitor of USP4 for 0 hours, 12 hours, 24 hours, 48 hours, then collecting cells respectively and the expression of USP4 and TβRI and Smad7 protein was detected with Western blot again;(3) HSFB whose density is 1×104 per hole was vaccinated in 96-well plate to culture for 24 h, then they were intervened by 5μmol/L Vialinin and The MTT method was to testify the proliferation of HSFB and The cells unintervened were regarded as the control group.Results:1. Immunohistochemistry showed that USP4 in NS has a small positive expression in the epidermal basal cell and hasn’t an obvious positive expression in FB.In addition to the epidermal basal cells, USP4 has a large amount of positive expression in fibroblast cell membrane and cytoplasm of HS and they were significant difference(P<0.01); TβRI hasn’t been shaded in the fibroblastof NS.However, TβRI has a lot of positive expression in the fibroblast cell membrane and cytoplasm of HS, which has a significant difference compared with the NS(P <0.01).2. As showed in Western blot, USP4 and TβRI protein in hypertrophic scar tissue were high expression and they were low expression in normal skin tissue and both comparison had a significant difference; USP4 and TβRI in hypertrophic scar fibroblasts were higher than the normal skin fibroblasts(P<0.05), but Smad7 was a low expression in Hypertrophic scar fibroblast(P<0.05); After adding Vialinin A in HSFB, USP4 and TβRI protein expression reduced gradually in HSFB over time,especially in 12 hours decreased significantly(P<0.05), but Smad7 protein expression was increased gradually(P<0.05) and further confirmed USP4 are related to TβRI positively and negatively correlated with Smad7.3. MTT showed that the proliferative activity of intervened HSFB compared with unintervened HSFB was reduced(P<0.05).Conclusion:1. USP4 and TβRI protein increased obviously in hyperplastic scar tissue and cells, but Smad7 is lower in HSFB than NSFB.2. Down-regulation of USP4 expression in hyperplastic scar fibroblasts can significantly reduce the expression of TβRI and up-regulate Smad7 expression. It suggests possiblly that USP4 through regulating the TGF-β signaling pathways influences the formation of scar tissue.3. Down-regulation of USP4 expression in hyperplastic scar fibroblasts can slow proliferative activity of intervened HSFB and will be expected to provide a new idea for scar prevention. |
PDF Full Text Request