Proteomic Identification Of Differentially-expressed Proteins Involved In Carcinogenesis And Progression Of Human Colorectal Carcinomas And Clinical Pathological Study

Objective: The study aims to the screening of differentially expressed proteins among colorectal carcinoma tissues(with lymph node metastasis)/without lymph node metastasis) and the normal mucous epithelium by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry. We expected to find some potential proteins that play a essential role in carcinogenesis and malignant progression of colorectal carcinoma. These proteins might be useful in daignosis for colorectal carcinoma and as target proteins in gene therapy for colorectal carcinoma in the future. Methods: In sample preparation , the fresh carcinoma tissue was cut off from the specimen immediately after the specimen was resected from the body and then preserved in the -80°C refrigeratory. The normal mucous tissue was cut off at the same time at the location far from the tumor over than 15cm. The tissues were classified into three groups according to the postoperative pathological diagnosis : carcinoma tissue with lymph node metastsis, carcinoma tissue without lymph node metastsis, and normal mucous tissue . In the first part, protein separation was performed by two-dimensional gel electrophoresis(2-DE) with immobilized PH gradient(IPG) strips.After silver-staining, the images of the gels were acquired by the scanner and then analyzed by using PD-quest7.3 software to find the differentially expressed protein-spots in each group. Further the differentially expressed protein-spots was incised from the gels and digested by trypsin . We acquired the peptide mass fingerprintings (PMF) by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and identified the proteins by data searching in the Mascot-database. Next, part of diffirentially expressed proteins including glutathione S-transferase(GST), heat shock protein27 (HSP27), liver-fatty acid binding protein(LFBP) were assayed by Western blot. In the second part, colorectal carcinoma associated tissue chip including colorectal carcinoma tissue, normal mucous tissue, and adenoma was established, GST and HSP27 expressions were assayed tissue chip by immunohistochemical method.