The Research About Effects Of SARP1 On Hypertrophic Scar Fibroblasts And Its Regulating Mechanisms

Background: Fibroblasts are the most effient cells during tissue repair. The apoptosis of fibroblasts usually appeares inhibited upon the formation of hypertrophic scar. However, the mechanisms of apoptosis inhibition are still unclear until now. Any new knowledge on signal pathway of fibroblastic apoptosis regulation would be beneficial for clinic treatment. Using DNA Microarray, we discoveried an anti-apoptosis gene, SARP1, which expression was pronounced high at early stage of hyperstrophic scar. These results indicated that SARP1 could be a key regulator for hyperstrophic scar. How does SARP1 work and what are its interactive proteins? Our research focused on these questions.Objection: To explore the effects of SARP1 on hyperstrophic scar fibroblasts (HSFb) and its regulating mechnisms. To find out the interactive proteins of SARP1.Methods:1. DNA fragment of SARP1 was amplified using RT-PCR and cloned into the vector based on adenovirus. The recombinant vector was indentified by enzyme digestion analysis and sequencing, and then packaged into 293FT cell line using liposome-transfecton method to produce adenovirus.2. After adenovirus infection, the expression of SARP1 in HSFb was confirmed by RT-PCR and western blot.3. The effect of SARP1 on proliferation of HSFb was detected by MTT assay, and the effect of SARP1 on apoptosis of HSFb was detected by annexin V-PI staining.4. The interactive proteins of SARP1 were separated using immunoprecipitation and proteomics. They were then indentified by mass spectrometry (LC-ESI-MS/MS).Results:1. Recombinants were confirmed using restriction enzymes and DNA sequencing. After adenovirus infection, both protein and mRNA of SARP1 were detected in HSFb. 2. Measured by MTT assay, the proliferation of HSFb was positively regulated by SARP1 (P<0.01).3. The apoptosis of HSFb was inhibited by the expression of SARP1 comparing to control group (2.86% vs 8.03%).4. By immunoprecipitation, seven protein electrophoretic bands were found in SARP1-expressed HSFb. Ten proteins were indentified by LC-ESI-MS/MS method. We speculated that six of them might be the interactive protein of SARP1, which were periostin(OSF-2),PLAP-1,phosphoglyceratekinase 1,rCG50690,apolipoprotein A-I precursor and thioredoxin 1 (TRX1).Conclusions:1. A recombinant adenovirus vector containing SARP1 of human was successfully constructed.2. SARP1 could be highly expressed in HSFb by adenovirus infection.SARP1 exhibited the proliferation-enhancing and apoptosis-inhibiting effects in HSFb.3. Periostin(OSF-2),PLAP-1,phosphoglyceratekinase 1,rCG50690,apolipoprotein A-I precursor and thioredoxin 1 (TRX1) might be the interactive proteins of SARP1, through which SARP1 regulated the formation of hyperstrophic scar.