| Objective:In vivo and in vitro experiments were used to explore the changes and differences in radiosensitivity of residual cells and DNA damage repair of A549 and XWLC-05 cells after radiation,and related biological mechanisms.Methods:1.A549 and XWLC-05 cells were cultured in vitro,and the cells were irradiated with X-ray 20Gy,and the remaining cells proliferated and resumed stable growth,that is,the remaining cells after A549 irradiation(A549RT)and the remaining cells after XWLC-05 irradiation(XW-05RT).A549,XWLC-05 and A549RT,XW-05RT cells were irradiated with different doses of X-rays.Plate clone formation experiments were used to compare the differences and changes in radiosensitivity of cells in each group.2.20Gy X-ray irradiation of A549,XWLC-05 and A549RT,XW-05RT cells,0.5h,1h,6h,and 24h after irradiation,,using cellular immunofluorescence experiments to detect ?-H2AX reflects the cell DNA damage,Western blot experiments The changes and differences in the expression of DNA damage repair-related proteins and PI3K/Akt channel-related proteins were detected.3.A549 and XWLC-05 cells were inoculated into the right back of 5-week-old male BALB/C-nu nude mice to construct a nude mouse subcutaneous transplantation tumor model.When the volume of the subcutaneously implanted tumor in nude mice is about 0.5cm3,perform 6MV X-ray 20Gy single irradiation of the transplanted tumor,and the flow rate of A549 and XWLC-05 subcutaneously implanted tumor tissues at different time points after irradiation,When there was no difference in apoptosis and necrosis between the two groups of subcutaneous transplanted tumors,the nude mice were sacrificed,the tumor tissue was stripped,digested,and then inoculated into the right back of the new male B ALB/C-nu nude mice to construct a residual lung cancer transplanted tumormodel..When the tumor volume of nude mice reached 0.1 cm3,PI3K/Akt specific channel inhibitor LY294002 was intraperitoneally injected,and X-ray 20Gy was used to locally irradiate the transplanted tumors of nude mice to observe the growth of subcutaneous transplanted tumors and residual lung cancer transplanted tumors.Western blot experiments were used to detect the changes and differences in the expression of DNA damage repair-related proteins and PI3K/Akt channel-related proteins in subcutaneous and residual lung cancer tumors.Results:1.With the increase of irradiation dose,the survival fraction of A549 cells is higher than that of XWLC-05 cells;the cell survival fraction of the residual cells after radiation of A549 cells and XWLC-05 cells is lower than that of the mother A549 XWLC-05 cells,and the survival fraction of A549RT cells is higher than that of XWLC-05RT cells.2.The expression of y-H2AX in both the parent cell and the remaining cells after irradiation increased after 20Gy irradiation,and remained at a high level 24 hours after irradiation.At 0.5h,6h and 24h after irradiation,the expression of y-H2AX in A549RT cells was significantly higher than that in A549 parent cells(p<0.05);0.5h and 1h after irradiation,the expression of ?-H2AX in XWLC-05 residual cells was significantly higher than that of XWLC-05 cells(0.5hp=0.045975,p<0.05;1h p=0.004503,p<0.01),indicating residual.The degree of DNA damage of cells after irradiation was generally higher than that of parent cells.3.The expression of DNA-PKCs,Ku70 and Ku80 in A549RT cells was stronger than that of A549 cells at 6h after irradiation(p<0.01).The expression of AKT and p-AKT473 protein in A549RT cells was stronger than that of A549 at 6h after irradiation(p<0.05).There was no significant difference in the phosphorylation levels of p-AKT473 and p-AKT308 between A549RT cells and A549 cells after irradiation(p>0.5).4.The growth rate of XW-05 subcutaneous transplanted tumors was significantly faster than that of XW-05 residual lung cancer subcutaneous transplanted tumor group(P<0.05);the growth rate of tumors in A549 cell subcutaneous transp lanted tumor group was slightly faster than that of A549 residual lung cancer subcutaneous transplanted tumor group(p>0.05);the growth rate of subcutaneo usly transplanted tumors in the XW-05 group was faster than that in the A549 group(p<0.05).After treatment with LY294002,the volume of subcutaneously transplanted tumors and residual lung cancer transplanted tumors were significantly smaller than that of the untreated group(P<0.05)5.After LY294002 injection into the abdominal cavity of mice,the phosphorylation level of p-AKT473 in the transplanted tumor and residual lung cancer transplanted tissues of each group of mice decreased compared with before treatment(P<0.05),indicating that PI3K/AKT pathway activity was inhibited;combined with X-ray 20Gy irradiation Afterwards,the phosphorylation level of p-AKT 473 in XWLC-05 residual lung cancer xenograft tumor was significantly lower than that of XWLC-05 subcutaneous xenograft tumor group(P<0.05),and th e activity of PI3K/AKT pathway in XWLC-05 residual lung cancer xenograft tumor was significantly inhibited.There was no significant difference in the phosphorylation levels of AKT and p-AKT308 between XWLC-05 residual lung cancer xenografts and XWLC-05 subcutaneous xenografts(p>0.05).The expression of Ku70 in XWLC-05 residual lung cancer xenografts was significantly lower than that of XWLC-05 subcutaneous xenografts(p<0.05).After LY294002 combined irradiation,the expression of Ku80 in XWLC-05 residual lung cancer xenografts was significantly reduced compared to XWLC-05 subcutaneous xenografts(p<0.05),there was no significant difference in the expression of DNA-PKcs between the two groups(p>0.05).Conclusions:1.Different tumor cells have different sensitivities to radiotherapy,which may cause lung cancer to be heterogeneous to radiotherapy.2.The degree of DNA damage of the remaining cells after irradiation is increased compared with the parental cells after irradiation,and the DNA damage repair ability at the corresponding time point is enhanced.The difference between the two may affect the radioactivity of the cells3.Different lung cancer cells have different growth rates of subcutaneously implanted tumors.After radiation,the growth of residual lung cancer tumors slows down.PI3K/Akt channel inhibitor LY294002 significantly reduces the growth rate of tumors.4.The DNA damage repair of the two transplanted tumors has changed after radiation,which may be related to the activity of PI3K/Akt channel. |
PDF Full Text Request