Monitoring Gene Expression Profile Changes In Human Gastric Adenocarcinoma Using CDNA Microarray

Background:Gastric cancer is the most lethal cancer in our country. The etiology of gastric cancer is still unknown. Despite the increase in our knowledge of oncogenes and tumor suppressor genes associated with gastric cancer over the past decades, the mechanism of the process of multistage carcinogenesis is still unknown for gastric cancer. Wholly understanding of the changed gene expression patterns in the gastric carcinogenesis and screening the gastric cancer related genes would provide a credible key to improve the dignosis and therapy of gastric cancer.With the implementing of Human Genomic Project , the studies of gene expression and gene funtion will become the hot spot of life science. DNA microarray , which combined molecular biology and microelectronics.is the new DNA analysis and examining technology. DNA microarray has been widely applied in the gene expression now. Objectives:1. To Screen differentially expressed genes between human gastric adenocarcinoma and normal gastric mucosa using cDNA microarray.2. To make gastric adenocarcinoma related cDNA microarray slides.3. To study the expression of immunoglobulin light chain kappa and lambda (Ig K and Ig X ) in gastric carcinoma cell.Methods:1 .The clinic sample of gastric adenocarcinoma and matching normal mucosa were obtained from PL A general hospital of China. The tissues were verified with pathology. Total RNA was extracted from frozen sample using TRIzol reagent.2.cDNA microarray filters comprising 18000 clones were made by Max-Plank laboratory of ShangHai Institute of Cell Biology . mRNA was extracted from clinical samples with Qiagen mRNA Isolation kit. cDNA probe for hybridization to the microarray were made by reverse transcription of the purified mRNA in the presence of 33P-dATP . The labled cDNA pools were hybridizzed to the genefilter arrays in a roller hybridization oven . The filters were then exposed to a Pachard Phosphoscreen for 8h, and scanned in a Fujifilm ImageReader instrument. The signals resulting from the phosphorimageing of ImageReader were directly imported into the image analysis system of ArrayGauge .The image analysis system automatically locate, calculate , and store the intensity of cDNA spot from each array and simultaneously compare the two values of the same cDNA blot on the two different filters.To validate the results of cDNA microarray hybridization. The differentially expressed cDNA clones were subsequently analyzed by in situ hybridization and Immunohistochemical analysis(LSAB method). cDNA probe for in situ hybridization were labled by Bio-11 -dUTP.3. Fabrication of gastric adenocarcinoma related cDNA microarray slides.A set of cDNA microarray slides contain a duplicate set of 576 cDNA spots.These selected clones have been sequence verified, the PCR products of human genes were spotted onto a chemical-material-coated-glass plates in array. DNAs were fixed the glass plate after series of treatments.Both RNAs from the gastric adenocarcinoma and normal gastric mucosa werereversely transcribed to the cDNAs with the incorporation of fluorescent dUTP to prepare the hybidization probes. The mixed probes were hybridized to the cDNA microarray. After high-stringent washing, the cDNA microarray was scanned for fluorescent signals and showed differences between two tissuses.4. Using the Immunohistochemical technique (LSAB method) for detecting the expression of Ig K and Ig A in paraffin embedded tissues of human gastric carcinoma from 22 patients. Results:1. The samples which came from clinic gastric adenocarcinoma patients were pathologically dignosised and fresh enough to have high quality total RNA when the total RNA of the samples were verified in the agarose-gel electrophoresis.2. Six patients were screened for expresssion of 18000 clones in gastric adenocarcinoma and normal mucosa using by cDNA microarray filters. The genes whose ratios of T/N (gastric adenocarcinoma/ normal gastric mu...