Research On The Differentially Expressed Proteins Between Normal And Hypertrophic Scar Keratinocytes

The scar seems unavoidable during the course of wound healing.The incidence of pathological scar after any kind of wound healing is 5%-15%,that of post operation is 39%-68% and afer burn is 35-91%.So the scar is a kind of common pathologic impairment and the most freq. disease of plastic surgery.. But till now the pathogenesis of scar is not very clear and the therapeutic measures are insufficient. Former studies on scars mostly focused on the fibroblast to search the activitied pathogenesis of the fibroblast,but studies on the inhibit factors and reasons after wound healing were rare. Recently some studies and clinical phenomenons showed that epidermic cells ,especially keratinocytes may play an important role on turning the activitied fibroblast to an inhibited stage after the wound healing. The proteins synthesized and excreted by keratinocytes are key factors to perform the transforming functions. So to trace the special proteins is our final aim.Proteomics is becoming the most important scientific field after genomics. Although the concept of proteomics came up only several years ago, it has maderapid progress especially on the techniques. Now the major techintque of proteomics is two dimensional electrophoresis(2-DE) which can separate the proteins at high-flux. Proteomics also offers professional standard software to collect data and analyze the 2-D map.Then proteomics can also identify the protein spots by amino acid composition analysis, protein sequence analysis or mass spectrum to get the expression,transcription and post-translation decoration information of the proteins. Such are all useful tools for our study.Our study is based on the hypothesis that scar keratinocytes lose some functional proteins which can inhibit the proliferation of fibroblast. We use 2-DE technique to separate and compare the total ptrotein extract of normal and scar keratinocytes to find the differentially expressed proteins. Then we use matrix-assisted laser desorption/ionization time of flight mass spectrometry technique (MALDI-TOF-MS) to identify these proteins. At last we will assume the function of the identified proteins during the formation of the hypertrophic scar.Exp.1. The way of separating the keratinocytes and extracting the total proteinsObjective To grobe and establish a high-performance method to seperate the noncultured keratinocytes and extract the total proteins. Methods The epidermic tissues were separated from the dermic tissues by Dispase- II enzyme.Then the keratinocytes were separated by Trypsin from the epidermis.Proteins were extracted from keratinocytes and epidermic tissues by alternate freezing and thawing plus one-step clearage using lysis buffer. Results The most suitable concentration of Dispase- II enzyme and Trypsin is 0.2% and 0.25%. The most optimized prescription of the lysis buffer is 7M Urea, 2M Thiourea, 4%(w/v) CHAPS, 0.1M protease inhibitor and 2%(v/v)4-7IPG buffer. The highest purity of the extract proteins reached 4-6mg/mL ,that extracted by liquid nitrogen got a lower one under 2mg/ml. Conclusion It is feasible to digest and separate the noncultured keratinocytes by Dispase-II enzyme and Trypsin. The protein extract was fit for 2-DE analysis. Exp.2. The establishment and optimization of the hypertrophic scar keratinocytes' 2-D map.Objective To establish a suitable stage for the 2-DE analysis for hypertrophic scar keratinocytes to accomplish the differentially comparing groundwork. Methods According to the 2-DE protocol, try to find the optimized pH display range, isoelectrofocusing (IEF) protocol and high-performance separating gel's concentration for second dimension. Results We used 13cm, pH 4-7 IPG strip, boosted voltage with step and gradient at high voltage, used 12.5% separating gel(second dimension,thk=1.0mm)to get a optimized separating and display effect. Conclusion Proteins of the hypertrophic scar keratinocyte mostly concentrated at pi 4-7;using IPG phor can save a lot of time for IEF; using home-made gel-making device and 12.5% separating gel can get ideal proteins separating results. So the 2-DE stage for the hypertrophic scar keratinocytes was successfully established.Exp.3. The collection and analysis of the 2-D map image and the comparison of the differentially expressed proteins.Objective To trace the differentially expressed proteins between normal and hypertrophic scar keratinocytes and reveal the difference at quantity and quality. Methods We used ImageScanner to collect the 2-D map images and Melanie 3.0 software to analyze the images so that we could get informations from the map. Regarding the normal 2-D map from the same patient as the contrast ,also according to the data from the The Danish Centre for Human Genome...