Proteomic Analysis Of Mechanism Of Ageing In Human Normal Colon Epithelium

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.It is also the initiating link for many pathological changes and the location for most loci.Aging is usually defined as a process during which the normal physiological functions may gradually weaken along with the increase of age.It is a normal, complicated and irreversible biological process,and a comprehensive manifestation of the declined functions and physiological functional disorder in the body.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.is obviously increased.Aiming at the aging mechanism of colonic epithelium, many related studies have been carried out at home and abroad.Lopes,etc. observed the changes took place in the mitochondrial morphology and function of colonic smooth muscle cells in aged rats.Ono,etc.found,along with the increase of age,the DNA mutation frequency of epithelial cells in digestive tract was increased.In particular,the mutation frequency of colonic cells was the highest.Tsurudome,etc.found raised level of 8-hydroxydeoxyguanosine in colon carcinoma tissue of the aged,which suggested increased degree of injury on DNA of colonic epithelial cells by oxygen free radicals.Although previous studies have revealed the mechanisms of the ageing of the colonic epithelium and susceptibility of ageing colonic epithelium to cancer,most of these studies proceeded from certain or some genes.However, the ageing of colonic epithelium is a gradual complex process involved by multiple genes and multiple factors.Besides,these studies mainly centralized at DNA and RNA level.But due to the complexity of gene transcription and translation,changes in DNA and RNA cannot reflect the changes of protein completely.Besides,it is impossible to learn the modification information after the translation of protein,the interaction of protein,the dynamic changes of protein,etc.only by studies on genome.Traditional study and technology on a single protein cannot satisfy with the requirements of studying such complex vital phenomena as ageing in the postgenome times.But the emergence of proteome by taking intracellular entire protein as the object has provided new research train of thought and technology.Differential expressional proteome is an important part in proteome study.Different from expressional proteome(a method trying to understand whole intracellular expressional protein),study of differential expressional proteome pays more attention to differential expressional protein of cells under different physiological or pathological condition,thus to seek or find key protein under different physiological or pathological condition.So 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,which might provide significant clue for clarifying the molecular mechanism of ageing in the colonic epithelium.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 pattems 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 furtherly,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 expresion 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.