Proteomic And Genomic Analysis,Identification Of Differential Protein And Gene Expression In Human Primer Gastric Cancer

Gastric cancer (GC) is one of the most common cancers and a major cause of cancer death in our country. Histological studies have classified gastric cancer into two distinct groups, namely the intestinal (or differentiated) type and the diffuse (or undifferentiated) type. Surgery remains the standard primary treatment and the best palliation for patients with GC. However, most patients are diagnosed at advanced stage disease and the five-year survival rate is generally less than 10%, on the other hand, it is easy to recur after surgical resection.Therefore, the development of novel therapeutic modalities is an issue with great clinical importance. To achieve this goal, a comprehensive understanding of the complex mechanisms of gastric tumorigenesis will be essential. Molecular investigations have provided evidence the evidence that multi-step and multi-factorial alterations are involved in gastric carcinogenesis including activation of oncogenes(k-ras, C-Met, K-Sam and C-erbB2) and inactivation of tumor suppressor genes(p53, APC, DCC), together with the abnormal of mismatching repair gene and so on. However, above-mentioned genes changes do not occur in all phenotypes of GC, since the mechanism of different type of GC is diversity. Therefore it is necessary to analyze GC's gene expression profiles from genome-wide to systematically study gene variation in gene expression programs in GC to reveal the identity of genes involved in malignant transformation, progression, and/or metastasis. It provides the facility that the achievement of Human genome project(HGP) combined with the proteome research becoming a hot spot in the post-genome era. With the development of research, from analysis of the feature of cancer cell itself, scientists begin to use tissue specimen to study tumor's development andprogression seriously.Aim of the study: (1) To establish and optimize the two-dimensional gel electrophoresis (2-DE), and establish the computer assisted image analysis system for proteomics. (D To Screen protein patterns of human intestinal GC and compare differences in protein expression quantitatively and qualitatively, (3) To simultaneously investigate genomic and proteomic changes so that we can provide an global understanding of a lot of protein and gene difference in time and space to understand the mechanisms of GC comprehensively. (4)To look for possible mark of diagnosis and therapy.Methods: Two-dimensional (2-DE) polyacrylamide gel electrophoresis (PAGE) was applied which used immobilized pH gradients (IPG) as first dimension, vertical SDS-PAGE as second dimensional. A series of important factors, such as sample preparation, electrophoresis parameters, choice of IPG gels, concentration of SDS gels, protocol for staining were optimized to improve the resolution and the reproducibility. Using the proper method described above, the intestinal GC and noncancer mucosa protein were separated. The gels stained with silver were scanned by ARCUS II scanner, which generated the digitalized images. The difference analysis between GC and its counterparts was carried out by Melanie 3 software by using three pair of 2-DE maps. Two remarkably differentially expressed spots were selected to be incised from the silver-stained gels and submitted to in-gel tryptic digestion. The resulting peptide mixtures were analyzed by Matrix-assisted laser-desorption ionization time of flight massspectrometry (MALDL-TOF-MS) .High-throughout cDNA microarray technique was used to study the difference in gene expression between two types of GC tissue and their non-cancer mucosa counterparts. Pure mRNAs from total RNAs of 6 GC and corresponding non-cacncerous mucosa were reversly transcribed into cDNAs labeled with Cy5 and Cy3 dyes to make probe, then mixed and hybridized with the cDNA microarray consisting of 4096 gene, and scanned by ScanArray400 with uniform background for fluorescent signals.GenePixS.O software analyzed its difference. RT-PCR proved using 2 increased and 2 decrease genes in intestinal GC.Results: This study es...