The Expression And Significance Of SPARC Protein In Animal Model And Human Scar Tissue Fibrosis

Background: Skin scar is an unavoidable result of skin fibrosis after wound healing,surgery and burns.Excessive deposition of ECM(Extra Cellular Matrix)and imbalance between synthesis and degradation of collagen fibers are important pathophysiological processes of scarring.SPARC is a 43-k Da protein secreted from multiple cell types from the extra cellular matrix(ECM).SPARC is mostly expressed during the development of tissue remodeling processes like wound healing and fibrosis,as a collagen-binding protein,has shown effect both on collagen ECM assembly and TGF-?1 activity.In animal models and patients with fibrosis,higher expression of SPARC protein has been reported in many fibrosis diseases,such as Myocarditis,Pulmonary fibrosis,Glomerulonephritis,Cirrhosis of the liver,Scirrhosarca,and so on.During fibrosis,tissue remodeling caused by dysregulated secretion and deposition of ECM often refer to normal tissue function.SPARC expression and up-regulation has been found involved in all the process.Skin scar fibrosis,pulmonary and cardiac fibrosis and other fibrosis diseases have similar processes of collagen ECM assembly and TGF-?1 activity,but many fibrosis processes is regulated by SPARC,this inspire us to think about the relationship between SPARC and skin scar fibrosis.We use in vitro and in vivo studies to verify whether SPARC promoted formation of skin scar tissue.Methods1.Collecting the first affiliated hospital of **** in September 2015-September 2016 after about during the pathological scar tissue resection scar skin tissue and normal skin tissue specimens in the skin graft,and fixed specimens using immunohistochemicaltechnique,after testing in the scar tissue SPARC protein is expressed in normal skin tissue,different skin tissue was detected in SPARC synthesis and distribution of all patients with scar samples preoperative do not accept any scar treatment.2.Cultivating normal skin tissue fibroblasts and scar tissue fibroblasts,using exogenous SPARC protein trained,by deter mined by MTT method to detect SPARC fibroblast proliferation,after confirmed by TUNEL method SPARC inhibitory effect of cell apoptosis related,using Western Blot technique to detect exogenous SPARC protein function,the group of scar fibroblasts scar growth factors related to TGF-?1,MMP-2,expression of MMP-9.3.Establishing the model of rabbit ear hyperplastic scar line trace syringe HS locally injected my wife SPARC protein,comparing the experimental group and control group pathological scar in morphological differences,understanding of the different degree of hyperplasia of the HS SEI detection,immunohistochemical and Sirius red staining detection fibroblast proliferation and collagen and other extra cellular matrix deposition,Western Blot detection in blank control group HS and reagent group HS group TGF-?1,MMP-2 and MMP-9.To clarify the direct effect of SPARC protein on the promotion of pathological scar formation and its mechanism.4.SPSS18.0 statistical software was used to process experimental data,and IPP software was used for image analysis,data analysis,processing and mapping.*P< 0.05,**P<0.01,indicating that the difference was statistically significant.Results:Part I1.The scar tissue and normal skin tissue specimens were fixed and immunologic chemical,after the HPIAS-1000 high resolution color image analysis system to the dyeing result for image acquisition,detection of SPARC expression in the scar skintissue and normal skin tissues,thro?gh Ki67 staining showed that SPARC protein high expression in the scar skin tissues.Part II2.1 cell proliferation was detected by MTT,and it was observed that different concentrations of SPARC protein could promote the proliferation of normal fibroblasts and scar fibroblasts,and there was a significant difference between 10?g/ml and 100?g/ml and 200?g/ml of SPARC protein on cell proliferation after 48h(p<0.05).2.2 according to the detection of cell cycle detector,after SPARC protein treatment,fibroblast cycle increased from G1 to S phase,and the number of S cells was significantly higher than that of the control group.Cell apoptosis experiment also verified that the number of fibroblasts in apoptosis after SPARC protein treatment was significantly reduced.2.3 expression of SPARC protein was detected by Western Blot.There was a dose-effect relationship between SPARC protein and MMPs family and TGF-?1 in skin fibroblasts and scar fibroblasts.SPARC protein had a positive correlation with TGF-?1,which was contrary to the action trend of MMPs.Part III3.1 Preparation of rabbit ear hypertrophic scar model,and microsyringe ad mineistration of HS SPARC protein(observe and compare the difference between the experimental group and the control group in the general morphology of pathological scar;3.2 HI detection: different degrees of scar hyperplasia in rabbit ears were detected.Immunohistochemistry and Sirius red staining were used to detect fibroblast proliferation and collagen and other extra cellular matrix deposition.3.3 Changes in TGF-?1,MMP-2 and MMP-9 expression in HS tissues of the blank control group and the reagent group were detected by Western Blot,so as to clarify the promotion effect and mechanism of SPARC protein's direct effect on pathological scar formation.3.4 the promoting effect of SPARC on skin scar tissue is the same as the biological characteristics of tissue fibrosis.The biological characteristics used in tumor cells are different.Conclusion:1.SPARC protein was more highly expressed in human skin scar tissue than in normal skin tissue,and labeled SPARC protein was located in the cell matrix.2.The degree of proliferation of scar fibroblasts and normal skin fibroblasts co-cultured with SPARC protein increased,and the degree of proliferation of SPARC protein on scar fibroblasts was higher than that on normal skin fibroblasts.With the increase of SPARC protein concentration,the promotion effect became more obvious.3.Exogenous SPARC protein can stimulate fibrosis of skin tissue,increase fibroblast and collagen deposition,and has a stronger effect on scar tissue that has produced fibrosis than normal skin tissue;4.Exogenous SPARC protein can promote the high expression of scar positive correlation factors and the low expression of negative correlation factors after the intervention.SPARC protein has an effective effect on promoting scar fibrosis.