| Intestinal epithelial cells (IECs) are the major component of the intestinal mucomembranous barrier, and play important roles in the body owing to its many kinds of functions such as absorption, immunity, and secretion, and so on. However, IECs are sensitive to ionizing radiation (IR), and they are one of the main targets in case of whole-body irradiation. Although much success has been documented with pelvic cancer patients, certain side effects and complications have been encountered which limit its applications in cancer radiotherapy. Intestinal wound induced by large dose of IR can form, thus make the intestine lose its barrier function. This results in gastrointestinal syndrome including intestinal infection of endogenous bacteria, diarrhea and bloody stools. It is the main reason of high death rate of intestinal-type radiation disease. However, its etiology remains unclear. Some studies have showed intestinal epithelial can reconstruct to some extent after IR (non-lethal). This suggested survival IECs can differentiate and re-epithelize intestinal wound. It is possible to study the proteins related to injury and reconstruction of small intestinal epithelium irradiated by γ-Ray. With the accumulating evidence in the literature that new proteins are found to be implicated in radiation response, the molecular mechanism underlying the radiation response of the small intestine remains unknown.Our aim was to identify those proteins in the early-stage of irradiation injury. To achieve this purpose, we have chosen to use comparative proteome approach to identify mouse proteins whose expression is regulated by ionizing irradiation. First, we compare the proteome of normal intestinal epithelial cell line IEC-6 with that of irradiated one at 24h post irradiation. Then, the proteomes of sham irradiated mice and irradiated mice were compared at 3h and 72h post-irradiation. The differential displayed proteins were subject to MALDI-TOF-MS to establish identity. Furthermore, we confirm some differential proteins with the technique such as Western blot and RT-PCR. At last, we focus on the function of 2 proteins, which are up-regulated by ionizing irradiation. New clues are provided to understand the molecular mechanism of the intestinal type of radiation sickness. Main results are listed below.1. Establishment of experimental models It is reasonable and availble to use rat IEC-6 cell line as a vitro model. Dose and time-point was chosen based on our preliminary data of cell death rate and apoptosis rate under different radiation dose and time course. Our results demonstrated that a dose of 25Gy and 24hr post-radiation were suitable to study the proteome of ICE-6 cells. The extent of mouse intestinal injury post-radiation was dose-dependent. Our data showed whole-body radiation at a dose of 9Gy could lead to intestinal type of radiation sickness. Under this condition, the height of intestinal crypts and villi were unchanged 3h post-radiation. However, cell division of intestinal epithelial ceased, and intestine showed severely morphological injury. Further study showed the height of intestinal crypts was obviously increased 72h post-radiation, with villi decreased. This indicated many survival epithelial cells proliferated. 2. Compare the proteome of IEC-6 cell line post-irradiationIsolated total proteins of IEC-6 cells and irradiated cells, and separated them according to the guide-book of 2-dimensional electrophoresis. For isoelectric focusing (IEF), precast IPG strips were used. Samples were applied via rehydration of IPG strips in sample solution more than 12 hours. 500-1000ug of protein was loaded onto an 18cm IPG strip (pH3-10, linear), and isoelectric focusing was run for 32 KVh at a focusing temperature of 20℃. For the second dimensional separation, the concentration of homogeneous SDS-polyacrylamide gels was 13%. We separated the same sample for 3 times. After electrophoresis, the resolved proteins in the 2-DE gels were visualized by Coomassie blue R-250. The gels were scanned, and the ima...
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