ABSTRACT
Objective To establish differential protein expressing profiles of human gastric adenocarcinoma cell in primary or metastatic lymph node tissues by two-dimensional differential gel electrophorosis (2-D DIGE) so as to investigate the metastatic molecular mechanism of the gastric cancer.Methods After obtaining 6 samples of human primary gastric cancer and metastatic lymph node tissues,with manual no-staining frozen sections microdissection,human gastric adenocarcinoma cells from primary or metastatic lymph node tissues were isolated,and then the total proteins were extracted and purified.Highly sensitive 2-D DIGE was used to separate the total protein differentially expressed in the cells.The proteins were visualized by using a fluorescence scanner at appropriate wavelengths for Cy2,Cy3 and Cy5 dyes (Typhoon 9400).Image analysis was carried out with the DeCyder-Differential analysis software (Biological Variation Analysis version 5.0).Results Not only can the study procure defined adenocarcinoma cell populations from gastric primary or metastatic lymph node tissues,but also can resolve the problem of the change in 2-D DIGE patterns because of the varying in protein changes owing to dyeing.All these showed that the technique was simple,easy to perform,versatile and of particular usefulness when laser capture microdissection (LCM) was practically unavailable.The 2-D DIGE patterns with high resolution and reproducibility from adenocarcinoma cells in gastric primary or metastatic lymph node tissues were obtained.The number of spots in Gel1,Gel2 were 1 416 (similar 1 062,decrease 277,increase 77),1 299 (similar 1 050,decrease 157,increase 92),respectively.A total of 11 differential proteins were acquired by image analysis with DeCyder-Differential analysis software (Biological Variation Analysis version 5.0).Conclusions In this report,a simple,easy to proform method of protein epuration,manual no-staining frozen sections microdissection is described,and have used the highly sensitive 2-D DIGE for the identification of proteins differentially expressing in human gastric adenocarcinoma cells from primary or metastatic lymph node tissues.These results provide a fundamental basis for further study of metastatic mechanism of gastric cancer and screen its specific markers.