Proteomic analysis of mechanism of aging in human normal colon epithelium

Abstract

Abstract Human colon has the function of absorption and secretion, participates the regulation of water and electrolyte balance, and maintains the homeostasis. Colonic epithelial cell is one of the main target cells in the body, which is acted by causative agents and external environment. Gradual declination of the physiological function may also occur to colonic epithelium along with the increase of age. Meanwhile, the occurrences of various intestinal diseases, such as malignant tumor, etc. are obviously increased. Aiming at the aging mechanism of colonic epithelium, normal colonic mucous epithelial tissues of the young and the aged people were selected in this study to study protein which is related to the ageing of human colonic epithelium using comparative proteome technology and methods. Firstly, comparative two-dimensional gel electrophoresis (2-DE) technology was performed to separate the total protein of colonic mucous epithelial tissues of the young and the aged people, respectively. The well-resolved, reproducible 2-DE patterns of the total protein of colonic mucous epithelial tissues of the young and the aged people were established. Then, PDQuest software was used to analyze 2-DE images and 47 differential expressional proteins between the two groups were discovered. These differential expressional proteins were analyzed by both peptide mass fingerprint (PMF) and peptide sequence tag (PST) based on MALDI-TOF-MS (Matrix-assisted laser desorption/ionization time of flight mass spectrometry) and ESI-Q-TOF-MS (Electrospray ionization-quadrupole time of flight mass spectrometry). Among them, 38 differential expressional proteins were identified through searching SWISS-PROT protein database with Mascot software. In order to verify the results of comparative proteome study, real-time quantitative RT-PCR, Western blot analysis and immunohistochemical staining were used to determine the differential expressional levels of the partial proteins of colonic mucosa epithelial tissues of the young group and the aged group, and the results were identical with the proteome analysis. In order to verify the results of comparative proteome study further, a senescent model of NIH/3T3 cell was induced by D-gal, two proteins was analyzed by western-blot in the senescent cell. The result showed that the expression of EF-Tu and Rhodanese was declined in the senescent cell. This suggested the alteration could be associated with cell senescence in vitro. Above all, 38 differential expressional proteins between the two groups were identified by 2-DE in combination with MALDI-TOF-MS and ESI-Q-TOF-MS. These differential expressional proteins could be divided into nine main groups based on their functions: anti-oxidative proteins, proteins relative to signal transduction, proteins relative to apoptosis, chaperones, proteins relative to protein folding, metabolic enzymes, proteins relative to transcription and translation, proteins relative to energy production, cytoskeletal proteins. So the results of this study indicate that injury of mitochondrial function and decline of antioxidant capability may be important reasons for the aging of human colonic epithelium. These data provides useful new information for the study of the molecular mechanism of ageing of the colonic epithelium.