| Background and Objective: Laryngeal cancer is common in head and neck malignant tumor. Its high incidence keeps on rising. Once early diagnosing is made, the patients will get good results. Tumors always arise and develop accompanied by changes of tumor markers (TM) in very early time. By checking them we can detect tumor in initial period. However, no ideal TM is found for laryngeal cancer by now because most researches are unilateral or on the level of gene and mRNA. But it is proteins that really embody actual life. Proteomics is a new subject that study composing, expression, modification, and interaction of entire function proteins of special kind of cell or tissue at different time and different position. Although proteomic technique has been widely used to detect joint molecules related with tumor, it's just a beginning in laryngeal cancer field, which involves tissue and serum study. It is confirmed that there are proteomic differences between laryngeal cancer and normal tissue beside tumor, and also between diseased serum and healthy serum, but no symbol molecule of high specificity and sensitivity have been found out, even those differential proteins found out have not been completely checked and identified. As tissue proteins can best embody characters of tumor, and serum proteins are too easy to be influenced by disturbing factors, we choose laryngeal cancer and normal laryngeal epithelial tissue as objects to extract total protein, used 2-DE which is the only method to separate thousands of proteins on one glue, associate MS and bioinformatics to search and identify different proteins of these two kinds of tissue, aims at setting up a proteomic database of laryngeal cancer characteristic and roughly filter TM of laryngeal cancer. Method: ①Laryngeal cancer tissue and pair normal laryngeal tissue specimens were obtained from nine 43-65 years old, male glottis cancer patients in T1～2N0M0 period. All diseased tissue were diagnosed by histopathology in First Xiangya Hospital, Central South University as highly differentiated Squamous cell carcinoma. Normal epithelial tissue was from opposite side of larynx ventricle and false vocal cord which had been diagnosed by histopathology as healthy. We extracted total protein from fresh specimens and measured their concentration respectively with 2D Quant Kit reagent in order to make certain the amount need to administrate in electrophoresis. After that, the extractions were stored at -70oC. ②IPG-2D PAGE: Conducts were according to the manual described by manufacturer and Gorg. IEF was performed using IPG strip on IPGphor? Isoelectric Focusing System with totally 400μg proteins were administrated. After that, took out IPG glue quickly and made it equilibrium before entering Second-dimension SDS–PAGE electrophoresis. The protein spots were visualized by silver-based staining technique with the protein silver stain kit. The stained 2-DE gels were scanned on a scanner. In order to measure the reproducibility, 2-DE for the tumor and normal-tissues from the same patient was repeated three times, respectively. ③Analysis the images with PD Quest software. The differential protein spots were detected between laryngeal cancerous tissues and normal mucosa tissues. ④Selected 37 well-resolved differential spots, cut them from glue. Respectively bring them into decoloration, reversion, enzyme digestion, extraction, desalinization in turn, and then drop them on stainless steel plate. After dried up, they were analysed and identified by MALDI–TOF mass spectrometry. Then, we obtained their peptide mass finger printing (PMF). ⑤Recognized the apexes with Mascot distiller software, and search for database under certain condition. Results: ①We got clear, reproducible and well-resolved silver stained 2-DE patterns of human laryngeal cancer tissues and normal mucosa epithelial tissue protein expression. ②The image analysis in PDquest software showed that these 2-DE maps were reproducible. For laryngeal carcinoma tissues, a total of 1504±122 spots were detected, 1305±143 spots... |
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