Biological Basic Research Of Short-term Single High-dose Radiotherapy Mode

Objects:To compare the effects of different single doses of X-irradiation on human pancreastic cancer cell lines MIA PaCa-2 in order to build the stable biological model of radiation-induced apoptosis and explore the effect of X ray irradiation on cell cycle,determine biological mechanism of short-term single high-dose radiotherapy mode.Methods:MIA PaCa-2 cells were irradiated with X-ray at different doses of 0,2,5, 10,17Gy in vitro,light microscope was used to observe cell morphologic 24 hours after X ray irradiation;cells of each dose group were collected at different times after irradiation,A flow cytometry was used to detect the apoptosis quantitatively and analyze the change of cell cycle;cells of each dose group were collected after irradiation,Alkaline single cell gel electrophoresis were performed to detect DNA damage,The comet image were analyzed by CASP software to detect tail torque of each dose group;DNA gel electrophoresis was carried out to determine the electrophoretogram of each dose group for qualitative determination of apoptosis.Statistical analysis data were processed by SPSS13.0 software.Results:1,Light microscope observation:Compared to the 0Gy group,MIA PaCa-2 cells in other groups showed changes in morphology after X ray irradiation,cell's volume was enlarged,and cell differentiation appeared obviously;2,Cells apoptosis was detected by flow cytometry:The results showed that X-irradiation at each dose could induce apoptosis of MIA PaCa-2.In certain dosage range(0～10Gy),the apoptotic rate was increased with dosage increasing,the time appearing apoptotic rate peak was delayed with dosage increasing.When dose reincreased to 17Gy,the apoptotic rate was decreased and the apoptotic rate peak was ahead of time;3,Cells cycle was detected by flow cytometry:In certain dosage range(0～10Gy), the time appearing G2/M phase arrest peak was delayed with dosage increasing, which was earlier than the time appearing apoptotic rate peak;cells at each dose showed that G2/M phase arrest peak appeared,then G1 phase arrest peak appeared; 4,Alkaline single cell gel electrophoresis:The DNA damage extent of MIA PaCa-2 cell in 10Gy group was significantly higher than that in other groups(P＜0.05); The DNA damage extent of MIA PaCa-2 cell in 0Gy group was significantly lower than that in other groups(P＜0.05);DNA damage after X ray irradiation at each dose was dose dependent,10Gy group was the obvious one,17Gy group was relieved;5,DNA gel electrophoresis:The electrophoretogram showed from single DNA electrophoresis band to typical apoptotic ladder pattern with dosage increasing in certain dosage range(0～10Gy),as the dose increased further to 17Gy,irregular necrosis band appeared.Conclusions:1,Morphologic change of MIA PaCa-2 cell after X ray irradiation based on swelling;2,In certain dosage range(0～10Gy),the ideal model of radiation-induced time and dose dependent apoptosis could be established by MIA PaCa-2 cells radiated by X ray;3,X ray irradiation can cause G2/M phase arrest in cell cycles of this cell line,which conforms to the traditional radiobiology concept;4,Each dose of radiation could possibly induce the apoptosis of cells in different phases.It was suggested that there existed certain relations between the cells sensibility to X-irradiation and checkpoints;5,Cell apoptosis rate peak and G2/M phase arrest peak appeared earlier after 17Gy X ray irradiation,and cell cycle arrest was later than apoptosis rate peak,which can be explained by the oncosis as the major death way;6,Quantitative and qualitative studies on DNA damage suggested that the apoptosis is the major death way of MIA PaCa-2 cell after low-dose and middle-dose X ray irradiation while the oncosis is the major death way of MIA PaCa-2 cell after high dose X ray irradiation.