Comparative Proteomics Studies From The Serum Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is a human malignant tumor derived from nasopharyngeal epithelial cells, which is one of the ten highest incidences in our country and is the highest incidences of head and neck tumors. NPC is a secrete tumor and sometimes have lymph node metastasis or other organs metastasis before be found. So it is very important for therapy and prognosis of NPC patients to have early stage diagnose. But we have no early stage diagnose method of NPC until now. Therefore, it is very important to study NPC associated serum proteins by comparative proteome analysis.The measurement of the novel NPC associated serum proteins can be an effective clinical access for detecting, diagnosing and monitoring the disease and path physiological processes.In our study, we collected 37 sera from nasopharyngeal carcinoma patients whose have lymph node metastasis, 27 sera from nasopharyngeal carcinoma patients whose have no lymph node metastasis and 42 sera from normal individual, pooled these samples into three groups by two-dimensional polyacrylamide gel electrophoresis (2-DE) we established at first. After verified in randomly selected individual samples, several different proteins were screened by software analysis, picked out, digested in gel and identified by mass spectrometry. So we identify some proteins associated with NPC and some proteins associated with lymph node metastasis, and validate the proteins associated with NPC by ELISA test and immunohistochemistry test.Results were as follows:1. In the tests of set up serum 2-DE techniques, Various conditions of 2-DE of serum were adjusted and optimized, such as how to collect and save serum sample, how to depletion high abundance proteins of serum, how to control the conditions of electrophoresis and the quantum of serum sample, and how to select IPG dry strip and confect buffer solution. A steady and high repetition 2-DE technique which can enhance resolution effective was established. And more protein dots especially some low abundance proteins were separate.2. In the tests of NPC serum comparative proteomics, from optimized 2-DE gel images, twenty-nine spots were differentially expressed and identified by MALDI-TOF-MS to be twenty-three proteins. Over all, transferrin, ZNF544 protein, transthyretin, FAD-synthetase and NM23-Hlprotein were under expressed and 12-lipoxygenase, serum amyloid A1 protein precursor, Cytochrome P450, sICAM-1, Cathepsin G and Lysine-specific histone demethylasel were over expressed in two NPC groups compared to healthy group. But 12-lipoxygenase, sICAM-1, Cathepsin G and Lysine-specific histone demethylasel were revealed in the group of NPC patients with lymph node metastasis compared to the group of no lymph node metastasis, but heat shock protein 70 expressed significantly and constantly only in NPC patients with lymph node metastasis group.3. In the tests of validate the proteins associated with NPC by enzyme-Linked immunosorbent assay (ELISA), the levels and positive rates of serum sICAM-1,SAA and HSP70 of NPC group were significantly higher than those in the normal subjects group and there have significantly difference between the NPC group of lymph node metastasis and the group of no lymph node metastasis. The 3 proteins became even higher as the malignancy of NPC progressed. And the 3 proteins have pertinence each other by bivariate analysis. The positive rates of ICAM-1 and HSP70 of NPC group of IHC tests were 75.4% and 63.2%. The two proteins have pertinence with TNM stage of NPC. And the positive rates of IHC of ICAM-1 and HSP70 in the NPC group of lymph node metastasis were significantly higher than those in the NPC group of no lymph node metastasis. We thank the two proteins were significantly in estimate clinical TNM stage and prognosis of NPC patient.In conclusion, in our study a good method of 2-DE technique for serum was established after long explorations and test, witch can be effectively applied in serum proteomics research. We also identified 23 serum proteins associated with NPC and validated thereinto 3 proteins, which might be useful to early diagnose and therapy of NPC. And our progeny might provide clues for further elucidating the role of these proteins in NPC carcinogenesis and developing potential associated biomarkers.