| Objective: Esophageal cancer is one of the common malignant tumor ofthe human, has posed a grave menace to the health of human beings. China isa country with high-incidence of esophageal cancer, it，s morbidity andmortality are the top of the world. Esophageal cancer is lack of diagnosis andtreatment methods, and with poor prognosis. If want to effectively improve theprognosis of patients with esophageal cancer will solve early detection, earlydiagnosis and early treatment. Therefore early diagnosis of esophageal canceris the hot issues in the research of esophageal cancer, only to solve the earlydiagnosis of esophageal cancer, will be able to improve the treatment effect ofesophageal cancer，improve the5-year survival rate for patients after theoperation. Proteomics as a new platform for cancer research, through thestudy from the overall level of protein network control, is significant in thedevelopment of early diagnosis of esophageal cancer, search for new markers,as well as the development of novel targets for drug treatment, etc.Metallothionein(Metallothioneins, MTs) is found for the first time in thehorse’s kidneys in1957. Is a kind of low molecular weight (6-7kDa), rich incysteine (30%), lack of aromatic amino acids, with the maintenance ofmetalion homeostasis, detoxification of heavy metals and the free radicalscavenging effect. People know it as the most effective free radical scavenger,can suppress cancer, eliminate the role of inflammation, prevention andtreatment of kidney disease and treatment of gastric ulcer. Its specialstructure、 unique physical、 chemical properties and biological functionsplays an important role for the research and study in the occurrence anddevelopment of the disease. Present, there are lots of research reports aboutMT protein in esophageal cancer and adjacent normal tissue, while theresearch about MT protein related protein is less, find and screening the protein between esophageal cancer tissue and adjacent normal tissue specificMT protein related protein is very important for the early diagnosis ofesophageal cancer. This study used immunoprecipitation(co-immunoprecipitation, Co-IP) and SDS-polyacrylamide gel electrophoresis(SDS-Polyacrylamide gel electrophoresis, SDS-PAGE) technique combinedwith liquid chromatography mass spectrometry (Liquid chromatography-massspectrometry, LC-MS) technology, screening and identification of differencesspecific MT protein related protein between esophageal cancer and adjacentnormal tissue, to find the protein with closely related to the occurrence anddevelopment of esophageal cancer, through the further in-depth research topromote the solving of early diagnosis and treatment for esophageal cancer.Methods:1Select esophageal cancer tissue and normal esophageal tissue from14patients that had10males and6females, with the age between42to76yearsold and among them the number of upper, middle, and lower part of theesophagus were2,8,and6. There were2stageⅠcases,5stage IIA cases,4stageIIB cases,4stageⅢcases, and1stageⅣcase and there were eight cases in welldifferentiated carcinoma,54cases in moderately differentiated carcinoma, and6cases in poorly differentiated carcinoma. These petients were operationed inThoracic Ward of the Fourth Affiliated Hospital of Hebei Medical Universityfrom August2011to February2012. Select tumor tissues by operations andnormal esophageal tissues which were more than5cm away from the cancertissues. All the processes were done in a low temperature environment, andafter the collection and registration, tissues were invested in liquid nitrogentank to-80℃refrigerator freezing. Tissues those postoperative pathology wasesophageal squamous cell carcinoma were taken in the study.2Extract total soluble protein from these tissues and test theconcentration of protein samples using the Bradford method. We separate theproteins by co-immunoprecipitationand SDS-PAGE， then compare the gelelectrophoresis of the Normal esophageal tissue and esophageal cancer tissuesso as to find and excise the different protein bands. 3We dissolve the different protein bands filtered in the gel, analyze thedifferentially expressed proteins extracted by LC-MS and then identify theprotein by biological information database.4We select and verify one of the differentially expressed proteins whichwere identified by Western blot on protein expression levels in order to testwhether it maintains consistency with the proteomics test results.5Statistical analysis: Use Fujifilm Las-4000Statistical analysis softwareto analysis integral optical density in the band which we get, with β-actin asinternal reference. Then use software SPSS13.0statistics processing line andsingle factor analysis of variance between groups. All the test results recordedaccording to the mean and standard deviation (x±SD), and EXCEL mapping.Results:1We use the method of Co-IP and SDS-PAGE get the MT relatedprotein gel of esophageal cancer tissue and normal esophageal tissue. Select64different bands which are high abundance and structural protein bands, cutand dissolve them. We identify31proteins by LC-MS. We found three kindsof protein maybe related to the occurrence and development of esophagealcancer. They are Lysozyme C, zinc-alpha-2-glycoprotein and lipocalin-1isoform1.2Using Western blot technique to validation lysozyme C in protein level,the results showed that compared with the normal esophageal tissue the MTstrip contains lysozyme C are significantly enhanced(t=-32.183，p=0.000),which we obtained the same results in proteomics.Conclusion:1Form the experiments we found that3kinds MT related differentiallyexpressed proteins in esophageal cancer tissue and normal tissue, andconfirmed that these MT related proteins are expression differences inesophageal cancer tissue and normal tissue. These three proteins may beinvolved in the occurrence, invasion and metastasis of esophageal cancer.2Using Western blot technique to validation lysozyme C in proteinlevel, it showed the same results with the research method of proteomics. It proved that our results have higher credibility.3Lysozyme C have high expression in esophageal cancer tissue than innormal tissue, prompt that the high expression of lysozyme C is closely relatedto the occurrence and development of esophageal cancer.The function studyof lysozyme C may provide the theoretical basis for the research of occurrenceand development in esophageal cancer, and may provide new targets for thetreatment of esophageal cancer. |
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