The Role And Mechanism Of SIP1in Hypertrophic Scar Formation

【Background】Hypertrophic scar is a fibrosis condition with the deep dermal tissue caused byvarious reasons, which results into the local fibroblast proliferation and collagen synthesisincrease. A large number of studies have shown that numerous of cytokines were involvedin wound repair process. Transforming growth factor-β1（TGF-β1）is play a key role in thescarring, which can be mediated by a variety of functions of cells. Activities of TGF-β1ismainly mediated by intracellular downstream molecule named as Smads. Smad proteinswithin the cell nucleus can regulate of gene expression by interaction with manytranscription factors. SIP1(Smad interacting protein1) can antagonism TGF-β1bycombination with Smads protein. SIP1also can inhibition the transcription of collagengene by combination upstream gene. Kato M found that the SIP1expression is low andrelated to the renal fibrosis mediated by TGF-β1pathway. In our previous study, the SIP1expression were lower in the hypertrophic scar tissue compared with normal skin tissues in the mRNA level. However, the specific mechanism of SIP1in hypertrophic scar is notclear, so this study will be further clarity the effects of SIP1on molecular mechanism ofhypertrophic scar formation, and provide a new theory for the prevention and treatment ofhypertrophic scar.【Purpose】1. To verify the SIP1expression in the hypertrophic scar tissue and the hypertrophic scarfibroblasts.2. To elucidate the role of SIP1in hypertrophic scar formation; validated by genetransfection experiment in vitro, and rabbit ear scar model in vivo.【Content】1. The expression of SIP1in hypertrophic scar tissue is detected by theimmunohistochemistry and immunofluorescence method. The expression of SIP1inhypertrophic scar fibroblasts is detected by immunocytochemistry.The expression ofSIP1, COLⅠA2and-SMA in the hypertrophic scar tissue is detected by Westernblot detection.2. Normal skin fibroblasts stimulated with TGF-β1and the expression of SIP1, COL ⅠA2and-SMA is detected.3. The expression of SIP1and fibrosis-related factors-SMA, COL Ⅰ A2, COL Ⅲwere detected by build adenovirus-mediated SIP1（Ad-SIP1） and transfectionhypertrophic scar fibroblasts. The expression of phosphorylation Smad2（P-Smad2）and phosphorylation Smad3（P-Smad3）were detected. The effects of Ad-SIP1on scarcollagen fibers is observed by construction rabbit ear scar model.【Results】1. The immunohistochemical detection results show that compared with autologousnormal skin tissue, the expression of SIP1in hypertrophic scar tissue were reduced.2. Immunofluorescence test results showed that compared with autologous normal skintissue, SIP1in the hypertrophic scar tissue were low expression. 3. Immunocytochemistry test results show that compared with the autologous normalskin fibroblasts, the expression of SIP1in hypertrophic scar fibroblasts were lowexpression.4. The results of Western-blot show that compared with autologous normal skin tissue,the expression of SIP1were lower in the hypertrophic scar tissue, fibrosis-relatedfactors COLⅠA2and-SMA expression were higher.5. TGF-β1stimulated normal skin fibroblasts, detection by Western-blot, results showthat the expression of SIP1gradually reduced; the COLⅠA2and-SMA expressionwere increased.6. After Ad-SIP1transfection of scar fibroblasts, the results of Western-blot show thatthe expression of-SMA, COLⅠA2, COL Ⅲ were obviously lowere than controlgroup. When cells given TGF-β1stimulation, expression of-SMA, COLⅠA2, COLⅢ in each group were raised, but express levels of Ad-SIP1+TGF-β1groups wereremained lower than control+TGF-β1group.7. Western-blot test results show that after Ad-SIP1transfection of scar fibroblasts, theexpression of P-Smad2and P-Smad3were obviously lowere than control group.When cells given TGF-β1stimulation, expression of P-Smad2and P-Smad3in eachgroups were raised, but express levels of Ad-SIP1+TGF-β1groups were remainedlower than control+TGF-β1group.8. In order to verify the results of in vitro experiments, we construction the model ofrabbit ear hypertrophic scar. After injection Ad-SIP1in the rabbit ear hypertrophicscar, by Masson staining showed that normal rabbit skin collagen more rarefaction,arranged layered, dyeing with pale blue; in the control group, PBS treatment groupand negative contro（lAd-NC）group show that the dermis were thicker, collagen fiberbulky density, orientation disorder; in Ad-SIP1group show that rabbit ear scarcollagen fiber density decreased, arrangement direction were orderly.【Conclusion】1. Compared with autologous normal skin tissues and its derived fibroblasts, the expression level of SIP1in hypertrophic scar tissue and its derived of hypertrophicscar fibroblasts were lowered significantly.2. TGF-β1stimulate normal skin fibroblasts, induced expression of COLⅠA2and-SMA, inhibit SIP1expression at the same time.3. Overexpression SIP1in scar fibroblasts by gene transfection technology that cansignificantly inhibit expression of fibrosis-related factors-SMA, COL Ⅰ A2andCOL Ⅲ; the molecular mechanism related to downregulation of P-Smad2andP-Smad3.4. Build the model of rabbit ear hypertrophic scar in vivo experiments, give rabbit earscar exogenous gene（Ad-SIP1） therapy that decrease the thickness of the collagenfiber and improve its organizational structure.