Proteomic Study Of Secretory Proteins By Fibroblast From Both Hypertrophic Scar And Normal Skin Tissues

Hypertrophic scar is a skin fibroproliferative disorder, which usually happen after serious skin trauma and extensive burn. Hypertrophic scar not only deforms the appearance of the patients, but also severely affects the psychological health and body functions. Hypertrophic scar characterizes excessive deposition of extracellular matrix (ECM).Because of most of the experimental animal such as rat, mouse, rabbit and pig cannot form hypertrophic scars, it's very difficult to study hypertrophic scar directly.Previous study demonstrated that, compared with fibroblasts from normal skin, fibroblasts from hypertrophic scar secreted more ECMs(typeⅠandⅢcollagen), versican, biglycan and other growth factors(such as TGF-βand insulin-like growth factor) and less remodeling enzymes, including collagenase and other matrix metalloproteinases, and small proteoglycan decorin, which plays a very important role in preventing scar formation. So fibroblasts play a key role in scar formation.Self-secreted cytokines are very important to cells themselves, which can affect the proliferation, differentiation, migration and other physiological functions. So in this experiment, we chose culture supernatant of fibroblasts derived from both hypertrophic scar and normal skin tissues as our study object. We established the secreted protein profiles of both cells and found out that protein vasorin and procollagen C endopeptidase enhancer 1 were highly related with scar formation.Methods:1. Isolation of fibroblast from both normal skin and hypertrophic scar tissues: the normal skin and scar tissues were obtained from Southwest Hospital, and enzyme digestion method was used to isolate the fibroblasts.2. Collection and separation of the supernatant proteins: After six day culture with serum-free media, the culture supernatant was collected. Ice acetone was used to precipitate the proteins in the supernatant. After centrifugation at 12000rpm about 30min, the supernatant was discard. The obtained proteins were dissolved in lysis buffer and 10% SDS-PAGE was performed to separate the protein.3. In-gel digestion:In-gel digestion was performed as follows: the SDS-PAGE gel was cut into gel slices, each of which contained a stained protein(s). The resulting slices were destained at 37°C in 50mM NH4HCO3 containing 50% ethanol. After the dye (Coomassie Brilliant Blue) was completely removed, the slices were washed with 25mM NH4HCO3 (pH 8.0) and dehydrated with acetonitrile (ACN). After being dried in a SpeedVac concentrator, the gel-bound proteins were reduced in 10mM DTT and then alkylated in 55 mM iodoacetamide (IAA) containing 6M guanidine hydrochloride. The gel pieces were then washed with 25 mM NH4HCO3, and again dehydrated with ACN and dried in a SpeedVac. The dry gel pieces were reswollen with 25μL of 25mMNH4HCO3 containing 0.5μg of trypsin and 0.1% n-octyl glucoside (W/V). Digestion was carried out at 37°C overnight. The peptides were extracted two times with 50μL of 5% formic acid and 50% acetonitrile by sonication for 15min. The combined extracts were evaporated to about 2μL in a SpeedVac and stored at -80°C.4. MS identification: The parameters for the mass spectrometer: the skimmer voltage was set at 40.0 V; the Cap Exit at 250.2 V; the Oct1 DC at 12.0 V; the Oct2 DC at 5.62 V; the Trap Drive at 85.0; the Oct RF at 216.3 Vpp; Lens 1 was set at–5 V and Lens 2 at–60 V. Data were analyzed using Spectrum Mill proteomics software (Rev A.03.02.060; Agilent).Raw data were extracted under default conditions and searched against mammalian sequences in the swiss-prot database using trypsin as the protease, allowing 2 missed cleavages, and including variable modifications of oxidized methionine and N-terminal glutamine conversion to pyroglutamic acid in the search. Peptides were considered valid with a forward–reverse score >2 and a rank 1–rank 2 score >2, a score threshold >7.67, and percentage-scored peak intensity >70%. Only proteins with 2 or more validated peptides and a total score >25 were considered valid for reporting. To compare identified proteins between treatment groups, the number of spectra and the summed ion intensity of peptides for each protein (total ion intensity) were used as indicators of protein amounts. Because these are semiquantitative metrics, we only considered those proteins with at least 5 additional spectra and at least a 10-fold increase in total ion intensity sufficiently different for reporting. To evaluate signs of matrix degradation, gel slices were used to estimate the molecular weight of proteins using the molecular weight marker as well as the molecular weight of some of the protein constituents.5. Analysis of the identified proteins: GO annotation was performed to select out the serected proteins and extracellular matrix proteins. Then proteins related to scar formation was picked out and analyezed.6. Retrospective verification: Western blotting and immunohistochemistry was applied to verify the proteins that were related to scar formation.Results:1. 82 and 79 proteins secreted by fibroblast from normal skin and scar tissues were identified respectively. Among these proteins, 43 and 37 proteins were annotated by GO as extracellular region proteins respectively. Further analysis showed that 25 proteins were secreted by both cells, 18 proteins secreted by normal skin fibroblast and 12 secreted by hypertrophic scar fibroblast specifically.2. We analyzed the function of these proteins by GO annotation. Most of the proteins secreted by both cells involve in binding function, account for 95.3% (41) and 89.2% (33) respectively. 9 proteins secreted by normal skin fibroblasts have signal transduction function and 9with structural molecule function, while proteins secreted by fibroblasts from scar tissues with such functions are 5and 10 respectively. 8 proteins form normal skin fibroblasts and 9 from the other cells have catalytic activity.3. We found that protein vasorin and Procollagen C-endopeptidase enhancer 1 secreted by fibroblast derived from scar tissues are highly related to scar formation.4. WB detection demonstrated that Procollagen C-endopeptidase enhancer 1 was highly expressed in culture supernatant of hypertrophic scar fibroblasts. Immunohistochemistry detection showed that Procollagen C-endopeptidase enhancer 1 was highly expressed in epidermis of normal skin. Conclusion:We established the secretory protein profiles of fibroblast from both normal skin and hypertrophic scar tissues. After comparison with that of firbroblasts from normal skin, we considered that protein vasorin and procollagen C-endopeptidase enhancer 1 were related to scar formation. Further WB detection confirmed our results. So vasorin and Procollagen C-endopeptidase enhancer 1 may be a new target for the treatment of scar.