| Objective:The study using X-ray irradiation gastric cancer BGC823 cells repeatedly and regularly,screen out the cell line BGC823 / IR that is radiation resistance and radiation tolerance,which is high energy X-ray ionizing radiation induced,using cell cloning experiments and Western blot technique to explore the radiosensitivity to X-ray of the gastric cancer BGC823 and BGC823 / IR.Method:(1)C ultured human gastric cancer cell line BGC823,when gastric cancer cell line BGC823 passage stablely and in good condition,send logarithmic phase cell exposed to the VARIAN linear accelerator 6MV,turn the X-ray irradiation dose rate to 200 c Gy/min,the source skin distance to 100 cm,the irradiation to 10 cm x 10 cm,1 cm equivalent tissue filler under the cell culture bottle,then give the gastric cancer cell line BGC823 2Gy dose X-ray.Put gastric cancer cell line BGC823 which has accepted 2Gy dose X-ray into cell culture incubator to cells in good condition,stable passage,and then repeat letting the logarithmic phase cells to receive 2Gy X-ray dose and so on.To the cumulative dose of gastric cancer cells BGC823 reached 2Gy,4Gy,6Gy,8Gy,10 Gy......30 Gy.Each group of different cumulative doses of gastric cancer BGC823 cells frozen specimens.(2)Using cell plate cloning experiments to explore the survival rate of gastric cancer BGC823 cell line without detection of X rays(control group)and gastric cancer BGC823 cell lines induced by X-ray(experimental group),then make the survival curve.(3)P-XRCC1 andγ-H2 AX protein expression in the cells of the 2 groups(gastric cancer cell line BGC823 and gastric cancer cell line BGC823/IR)were detected by Western blot method;To detect the expression of C leaved-caspase-3,PARP-1 andγ-H2 AX in gastric cancer BGC823 cells with different doses and different time intervals.Result:(1)C loning experiments show that 0Gy,1 Gy,2 Gy,4 Gy,6 Gy and 8 Gy X-ray,colony formation rate(PE,plate,efficiency)of control group and experimental group,were56.7%,44.2%,17.7%,4%,0.21%,0;53.6%,52.6%,26% 6.25%,1.53%,0.16%.(2)Western blot technology detect the P-XRCC1 expression to 4 Gy 6 MV X after irradiation 0h,3 h,6 h,9 h,control group respectively is 0.398 + 0.052,0.312 + 0.078,0.217 + 0.017,0.184 + 0.046,experimental group were 0.402 + 0.072,0.620 + 0.1,0.997 + 0.033,1.174 + 0.039.In normal control group,the expression of P-XRCC1 decreased gradually with the extension of time,and the expression of P-XRCC1 in the experimental group increased gradually with the extension of time,and there was a significant difference between the control group and the experimental group(P<0.05).The expression of γ-H2 AX in normal control group was 0.245 、0.748 、1.254 、1.468,respectively.The expression of γ-H2 AX in the experimental group were 0.262 、0.268 、0.711 、1.183,respectively.The normal control group,the expression of γ-H2 AX with time gradually increased,the expression of γ-H2 AX in experimental group with time gradually increased.However,the expression of-H2 AX in normal control group was significantly higher than that of the experimental group in the corresponding time period,and there was significant difference between the control group and the experimental group(γ-H2AX)(P<0.05).12 hours after treating with different dose,the expression of C leaved-caspase-3、γ-H2 AX of gastric cancer cell BGC823 increased with increasing dose,the expression of PARP-1 with higher doses decreased;the expression trend of C leaved-caspase-3,γ-H2 AX,PARP-1 of 24 hours is the same as the expression trend of 12 hours,but the expression level was significantly lower than the absolute value of the expression of 12 hours.Conclusion:(1)The results of cell clone test showed that the survival rate of BGC823/IR was higher than that of BGC823 cell line after irradiation with 6 MV X irradiation,suggesting that BGC823/IR cell line of gastric cancer may produce radiation resistance during X ray irradiation,and it may be related to cell repair mechanisms.The expression of P-XRCC1 in gastric cancer BGC823/IR cell line in the Western blot technology showed an increasing trend,while the expression of BGC823 cell line P-XRCC1 showed a decreasing trend,showed that the repair effect of BGC823/IR cell line was greater than that of BGC823 cell line,and the repair effect is stronger and stronger over time.The expression of γ-H2 AX in the BGC823/IR cell line was increased and the trend was less than that of BGC823 cell line,showed that the damage of BGC823/IR cell line induced by X ray was less than that of BGC823 cell line.The expression of C leaved-caspase-3、γ-H2 AX in gastric cancer cell BGC823 increased with increasing dose,the expression of PARP-1 decreased with the increased dose,suggesting that gastric cancer radioresistance may related to the apoptosis rate.(2)The production of radiation resistance gastric cancer cell line BGC823/IR may be related to the mechanism of cell repair. |
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