| Background:Adenoid cystic carcinoma of salivary glands(ACCs) also known as cylindroma, is a common dental malignant salivary gland tumors originated from epithelial tissues. It has biological characteristics such as strong local invasiveness, low regional lymph node metastasis rate, growth along nerves and blood vessels, distant blood-bone metastasis and so on. ACCs is of high malignancy and low radiation sensitivity. It is due to this that the tumor has a high relapse rate after surgical resection. If the methods of gene therapy can be introduced to improve the sensitivity of adenoid cystic carcinoma toward radiation, the effect of radiotherapy will be enhanced, and therefore the survival rate of patients may be brought up significantly. Because the activation of NF-κB signal transduction pathway has been proved to produce a major source of radiotherapy resistance, blocking this pathway by genetic engineering technology may help us improve the radiation sensitivity of adenoid cystic carcinoma.Objectives:The purpose of this study shall be:evaluating the expression level of P65 protein, which is a subunit of NF-κB signal transduction pathway, and detecting the apoptosis after the ACC-2 cells are irradiated by high-energy X-rays; exploring the relations between the P65 protein expressions and apoptosis; obtaining a series of images. These experimental data are bound to lay a good foundation for further radiosensitizing experiments.Methods:ACC-2 cell line was recovered from a liquid nitrogen jar, and the cells were subcultured for several generations to gain enough clones. After the cells turned into the logarithmic growth phase, they were irradiated by the high-energy X-rays of different doses, including 2Gy,4Gy,6Gy,8Gy, and 10Gy. In the next six time points of 1st,3rd,6th,10th,24th,48th hour after irradiation, the following four different experimental methods were used respectively to achieve the detection of NF-κB signal transduction pathway activity and the statistics of cell apoptotic rates. By immunocytochemistry, the time and spatial variability of P65 protein expressions in Adenoid Cystic Carcinoma cells of different radiation dose groups were observed, . with pictures captured by microscopic imaging system. The quantities of P65 protein in cells of each groups were semi-quantitively detected by the immunization protein blot (western blotting) technique in order to know the protein's expression patterns after irradiation. Flow cytometry was carried out to count the apoptotic rate of the irradiated cells at the six time points mentioned previously. Finally, TUNEL (terminal deoxynucleotidyl transferase-mediated nick end labeling) test was used to observe nuclear morphology of apoptotic cells, and image data were obtained by fluorescence microscopy imaging system. The environment in which the cells were cultured was strictly maintained. Control groups of cells without irradiation were set in each of the above experiments to make the results prominent. Some key steps were repeated twice in order to reduce systematic errors. Statistical test methods were used for data analysis.Results:1. It was observed by immunocytochemistry that in the control group cells P65 protein rarely appears in the cell nucleus and most of the protein was stained in the cytoplasm around the nucleus. After irradiated by the high-energy X-rays, more and more P65 protein went through the membrane and gradually concentrated in the central area of the nucleus. This process in cells can be accelerated by a higher radiation dose than a lower one.2. By means of western blotting, the quantity of P65 protein in total cellular protein content was found to be changing gradually as time went by. The protein expression in each cell group reached its peak after about 6 hours-10 hours. At the time point of the 3rd hour after irradiation, the P65 protein was found to have a higher expression with a higher dose irradiation correspondingly.3. Apoptotic curves were plotted according to the flow cytometry data of the 5 dose groups, which indicated that the apoptotic rate constantly changing at different time points, with the lowest points of apoptotic rates at the 10th hour for all the 5 dose groups after radiation. Also at the time point of the 3rd hour, the apoptotic rates were discovered to decline as the radiation doses increased. Compared with the outcomes of western blotting, the results indicated a negative correlation between the apoptotic rate and the P65 protein's expression (the data were statistically tested by Spearman method with SPSS 11.5, P=0.010<0.05)4. By immunofluorescence staining of TUNEL technique, the apoptotic cells were observed to have comparatively typical morphological changes in their nucleus, such as the chromosomes concentration, nuclear fragmentation and apoptotic bodies and so on, with the same trends in ratio of apoptotic cells as the data of flow cytometry.Conclusions:The expression of P65 protein in ACC cells is affected by the irradiation of high energy X-rays, which is dose-time dependent. After irradiated, the concentration site of P65 protein has a constant migration in the cells over time, and gets to the relative areas of gene regulation in the nucleus. In this migrating process, particular time corresponds to a specific position of P65 protein concentration. The quantity of P65 protein in total cellular protein content was found to be changing gradually as time goes by, which means that a particular protein quantity corresponds to a specific time point in a specific dose group of ACC cells after irradiation. If the dose of radiation increases, the process of the migration of P65 protein concentration site will accelerate and the quantity of the protein will grow slightly. It indicates a positive correlation between the dose and its effect. After irradiation, the different dose groups of ACC cells have their own characteristic apoptotic curves, and the apoptotic rates are also negatively correlated with expression levels of P65 protein.
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