| Oral lichen planus (OLP) is a chronic oral mucosal disease of long-termrecurrent. The World Health Organization (WHO) defined OLP as aprecancerous condition because of its chronic erosion and easy to becomecancer. The development and psychological factors of OLP are endocrinefactors, immune factors, infection and other relevant factors. The exactpathogenesis is not clear right now. Because of unknown mechanism, thetreatment has no significant effect. So exploring the mechanisms of oral lichenplanus become an urgent problem. In recent years, a new era of life sciencesappeared—proteomics ear. Proteomics is the second developed aresearch-based genome science. It is about the study of protein which by genetranscription product dynamics and on the overall level, the life can be directlystated the pathogenesis of physiological and pathological conditions.Proteomics is developing rapidly and two-dimensional gel electrophoresisseparation method is the most important. Two-dimensional fluorescencedifference in gel electrophoresis(2-D DIGE) is based on traditionaltwo-dimensional gel electrophoresis. It labeled the control group and theexperimental group with different fluorescent dye, and mixed together, thenperformed two-dimensional gel electrophoresis. Depending on the type offluorescent material excitation wavelength, it makes differentially expressedproteins in different colors on a2D gel images. It can detect a slight differencebetween the expression of protein samples and it is higher sensitivity,reproducibility and accuracy. Now this technology has been widely used inmedical research at home and abroad, especially in the application of thistechnique to identify cancer-associated molecular markers, so as to inhibit thedifferentiation of maligant cells, for the development of therapeutic drugstheoretical basis. But now at home and abroad the research reports are veryrare for the study of differentially expressed serum proteins from oral lichen planus patients by2-D DIGE. So this study screened and identifieddifferentially expressed serum proteins from oral lichen planus patients by2-DDIGE and move forward a single step to investigate the possible mechanismsof oral lichen planus.Objective：Identification of differentially expressed serum proteins werefrom oral lichen planus patients by two-dimensional fluorescence difference ingel electrophoresis(2-D DIGE) and Mass spectrometry. And the oral lichenplanus and health adult related proteins analysised to tell us where have beenchanced and which proteins may have a relationship with oral lichen planus. Itmakes early diagnosis of oral lichen planus, biological proteins targetedtherapy prognosis tends to be possible. Meanwhile it can also confirm2-DDIGE has practical value in oral lichen planus.Methods：1Comparision of two methods to extract human serum proteins：Twohealth adults were random selected and drawed veinal blood. Serum proteinswere cleaned by methanol,10mM ammonium bicarbonate and phenolextraction. Protein concentration was determined by Bradford method. Serumproteins were separated by SDS-PAGE and stained with coomassic blue,cleaned the coomassic blue, comparied the obtaining protein bands and weselect extraction method which is suitable for human serum proteins.22-D DIGE:6pairs of patients and matched health adults were randomselected and drawed veinal blood. Serum proteins were cleaned by phenolextraction and protein concentration was determined by Bradford method. Therepetitive of the proteins and the protein degradation were detected by SDS-polyacrylamide gel electrophoresis. Serum proteins were separated by2-DDIGE.2-D DIGE gels were scaned by Typhoon9410, and stained by DeepPurple，differentially expressed serum proteins were searched by differentialanalysis software version6.5,differentially expressed serum proteins whichwere greater than1.2were labeled and generated dig coordinates file, theproteins were diged with Ettan spot picker.3Differentially expressed serum proteins were identified by liquid chromatography-mass spectrometry: Protein-micelles were duged and cleaned,Trypsin was put into them, proteins were identified by LTQ XL enhanced twodimensional linearion trap mass spectrometer. To seach the possible peptide inorder to identify the possible differentially expressed serum proteins byBioworks Browser3.3.1with ESQUEST method.Results：1Comparision of two methods to extract human serum proteins andchoose the better one: Serum proteins were cleaned by methanol,10mMammonium bicarbonate and phenol extraction. Protein concentration wasdetermined by Bradford method. Serum proteins were separated by SDS-PAGE and stained with coomassic blue, cleaned the coomassic blue,comparied the obtaining protein bands. We found that the extraction methodwhich was cleaned by phenol extraction obtained the clearer protein bands andhigher degree of purification of the proteins. So phenol extraction method wasused in this study.2SDS-polyacrylamide gel electrophoresis: Each sample protein bandswere clear and higer reproducible.Protein loss was rarely to be found.32-D DIGE: Oral lichen planus serum and healthy human serum weremarked with different color fluorescent dye. In the2-D DIGE separation,sereral protein spots were overlapped in two colors, showing in yellow. Thisshowed the proteins in healthy individuals and OLP patients were at the samelevels of expression. While some specific expression showing red or greenshowed that healthy individuals and OLP patients were at the different levelsof expression. After6biological replicates, In average, the abundance of（583±183）serum protein spots was found in oral lichen planus patients.12highly reproducible spots were selected for mass spectrometry identification.Of these protein spots, the abundance of7proteins were consistent lower inoral lichen planus than that in normal adults. The abundance of5proteinswere consistent higher in oral lichen planus.4Differentially expressed serum proteins were identified by liquidchromatography-mass spectrometry: Identified12kinds of different proteins, respectively: Haptoglobin, Ceruloplasmin, Serum complement C3,Appolipoprotein A-Ⅰ, Kiniongen, Vitamin D, α-2-macroglobulin, Humanantithrombin Ⅲ, Secretory IgA, Serum complement component C9, Humanfactor B, β-1-metal binding globulin.Conclusions1Phenol extraction method is suitable for the extraction of human serum.2There were some differential proteins in serum from oral lichen planuspatients. In average, the abundance of（583±183）serum protein spots wasfound in oral lichen planu patients.12highly reproducible spots were selectedfor mass spectrometry identification. Of these protein spots, the abundance of7proteins were consistent lower in oral lichen planus than that in normaladults. The abundance of5proteins were consistent higher in oral lichenplanus.312kinds of different proteins were identified by mess spectrometry andin part of them Haptoglobin, Ceruloplasmin, Serum complement C3,Appolipoprotein A-Ⅰ, Kiniongen, Vitamin D, α-2-macroglobulin, Humanantithrombin Ⅲ, Secretory IgA, Serum complement component C9, Humanfactor B may related to pathogenesis of oral lichen planus. |
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