Bystander Effect And Transmission Effect Of Clinical Radiotherapy To Lung Adenocarcinoma A549 Cells And Normal Lymphocyte Cells

Background:radiation bystander effect refers to the non-irradiated cells showed the same or similar biology reaction phenomena as radiated cells, mainly including gene mutation,gene instability, DNA damage and chromosome break and mutation, cell proliferation and apoptosis, inflammation, tumorigenicity transformation and cancer formation, etc.During radiation therapy, in addition to the illuminated area of radiation injury symptoms, fatigue, pain, loss of appetite, indigestion, decreased white blood cell count and other systemic symptoms often happens in patients, and bystander effects may play an important role among them. The degree of distant organ damage is related with tissue types, and the non-irradiated cells can reduce irradiated cells’ damage. In clinical local irradiation, the overall radiation biology behavior will become more complex, and we need to jump out the traditional concept of direct illuminate damage and further understanding the mechanism of normal tissue damage.Objective:1.Using the 6 mv X-ray irradiated A549 cells’ medium as conditioned medium(ICM)to culture non-irradiated A549 cells, and researching non-irradiated cells’ bystander effect;2.Using the 6 mv X-ray irradiated A549 cells’ medium as the first generation conditioned medium(ICM) to culture non-irradiated normal lymphocyte cells, and researching non-irradiated cells’ bystander effect;Using fresh medium culture the above lymphocyte cells as the second generation medium to culture normal lymphocyte cells,and study those cells bystander effects.This research aims to provide a new train of thought to explain local leucopenia after irradiation, and helps to further understand the biological significance of radiation induced bystander effect.Methods:1.The A549 cells can be divided into the control group, the irradiation group,conditioned medium group, fresh culture groups. We select the A549 cells in logarithmic phase receive 0 Gy, 0.5 Gy, 1 Gy, 1.5 Gy, 2 Gy 6MV-X ray irradiation,and then transfer conditioned medium to non-irradiated A549 cells 3 hours later, clone formation method were used to detect cell survival fraction;Test apoptosis situation of A549 cells accepted 2 Gy 6 MV-X ray irradiation after 24 h, 48 h, 72 h, 96 h by using Annexin V- FITC/PI double dye flow cytometry;PI single dye flow cytometry were used to detect each A549 cells’ change of cell cycle after 24 h, 48 h, 72 h, 96 h.2.Healthy lymphocytes were divided into control group, the first generation conditioned medium group, the second generation conditioned medium group, the first generation intervention group, the second generation intervention group, control group using fresh culture. Use radiated A549 cells’ culture medium as the first generation group to cultivate normal lymphocytes 24 h, then remove lymphocytes from the first generation medium to fresh culture, then use the medium that cultivates cells after 24 h to deal with normal lymph cells the second generation conditioned group.The first and the second generation conditioned medium group both set up a set of dexamethasone intervention group, detect all experimental cells’ survival fraction and apoptosis rate after be cultivated in each medium for 24 h.Statistical processing:all the data is showed by mean ± standard deviation(mean+S.D.), use the independent sample t test, analysis all data by SPSS16.0 statistical software,, and consider data having statistical significance when p < 0.05.Results:1.The cell survival curve: cell survival fraction decreased with increasing doses after 0.5 Gy area. Compared with control group, the apoptosis rate in conditioned medium group increased with time increases, and reached the maximum at 72 h(p <0.05);G2/M phase cells increased significantly(p < 0.05)with time, and G2/M phase of the block is lifted after 72 h.2.The lymphocyte cell survival fraction of The first generation conditioned medium group and the second generation condition medium group decreased significantly than control group(p < 0.05), the apoptosis rate increased significantly(p<0.05);After dexamethasone intervention lymphocytes’ survival fraction increased than corresponding non-intervention group(p<0.05), the apoptosis rate significantly decreased(p<0.05).Conclusion:1.6MV-X ray irradiation can lead radiation induced bystander effect of A549 cells by conditioned medium, and that effect reached the maximum at 72h;2.Clinical radiation bystander effect can lead normal lymphocyte damage by conditioned medium, and this damage can result in healthy lymphocyte’s chain damage through the second generation conditioned medium, eventually produced a remote transfer effect.