| Purpose: Lung cancer not only has a high incidence,but also ranks first in mortality worldwide.For a long time,human life and health have been seriously threatened by lung cancer.Lung cancer usually appears in the form of pulmonary nodules in the early stages.In clinical diagnosis,pulmonary nodules are usually divided into benign and malignant nodules based on the size of the nodule diameter.40 % of them are malignant nodules.Adenocarcinoma accounted for 47 % of malignant nodules,squamous cell carcinoma accounted for 22 %,solitary metastatic lesions accounted for 8 %.Therefore,for patients with lung adenocarcinoma,timely detection,timely diagnosis and timely treatment have become the key to improving survival rate.Although there is no accurate and uniform definition of nodule diameter boundary size in different countries,it is currently considered important to detect and track solid nodules greater than 4 mm and ground-glass nodules greater than 20 mm.In 1990,Naidich and other scholars first proposed LDCT as a new method for lung cancer screening.In 2011,the United States carried out the lung screening test(NLST)for lung cancer patients,and regularly scanned the target population at low doses.The results showed that regular CT examinations reduced the mortality rate of lung cancer patients by 20 % compared with conventional chest X-ray photography.Later,American professional institutions proposed to use low-dose CT scans in clinical practice,and people at high risk of lung cancer should be screened regularly.In China,lung cancer screening was included in the ’ Rural Cancer Early Detection and Early Treatment Project ’ in 2009 and the ’ Urban Cancer Early Detection and Early Treatment Project ’ in 2012.After that,various regions have also carried out screening for high-risk groups of lung cancer in China.However,the radiation safety problems caused by CT scanning have long been concerned and controversial.The International Commission on Radiology said that for every l m SV increase in the dose of X-ray irradiation received by the human body,the probability of malignant tumors in the exposed person will increase by 1 /20,000.Although the radiation dose of a single single-site CT scan varies from a few to a dozen m Sv,which is much lower than the minimum radiation dose that may increase the risk of cancer-100 m Sv,radiation will accumulate in the body and still cause harm to the human body.The purpose of this paper is to explore the DNA damage of lung cancer cells after low-dose CT scan and the malignant transformation of lung cancer cells after long-term culture.Methods: Firstly,the irradiated cell model was constructed : A549 lung cancer cell line was selected and divided into three groups.The first group received a 3 c Gy irradiation.The second group received a 9 c Gy irradiation;the third group was continuously irradiated for 3 days,3 c Gy once a day.Unirradiated cells were used as control group.After irradiation,two time points of 6 hours and 24 hours were selected for detection,and then the cells were subcultured.The cells of 20 generations,40 generations and 60 generations were selected for detection.Western Blot was used to detect the expression of DNA damage-related proteins,cell cycle-related proteins,EMT marker-related proteins and metabolism-related proteins.Flow cytometry was used to detect the cycle distribution of irradiated cells.Colony formation assay and CCK-8 kit were used to detect the proliferation and cell viability of irradiated cells.Cell scratch test was used to detect the migration ability of cells after irradiation.Transwell assay was used to detect the invasion ability of cells after irradiation.In vivo,a subcutaneous xenograft model of nude mice was constructed.When the tumor grew and reached a certain size,the tumor-bearing mice were treated with 6 Gy X-ray irradiation or 5 μg/ml BLM(bleomycin).The tumor volume of the tumor-bearing mice was counted and the survival curve was drawn to observe the changes and differences in radiation resistance of tumor cells.The lung metastasis model of nude mice was established by tail vein injection of orthotopic tumor.The formation of metastasis in lung tissue of nude mice was observed by PET / CT and pathological section of lung tissue.Results: The phosphorylation level of DNA damage marker γ-H2 AX in cells was detected at 6 hours and 24 hours after irradiation.It was found that a single 3 c Gy and 9c Gy irradiation after 6 hours resulted in an increase in the expression level of phosphorylated γ-H2 AX in A549 cells.The highest level of phosphorylated γ-H2 AX was observed in the 9 c Gy irradiation group.3*3 c Gy fractionated irradiation did not cause a significant increase in intracellular phosphorylated γ-H2 AX levels.Compared with 6 hours,the expression level of phosphorylated γ-H2 AX was consistent in A549 cells irradiated with 3 c Gy and 3*3 c Gy 24 hours after irradiation.Flow cytometry results showed that the proportion of G2 phase cells in 3 c Gy,9 c Gy and 3*3 c Gy groups increased significantly compared with the control group at 6 hours and 24 hours after irradiation.Among them,the G2 phase arrest was the most obvious in the 3 c Gy group.At the same time,the expression levels of cell cycle-related proteins in each group were detected.The expression of P53 protein was increased in different irradiation groups,and the most obvious increase was the expression of P53 protein in3*3 c Gy group.The above results suggest that although the dose below 9 c Gy is small,irradiation of A549 cells also causes DNA damage,and at the same time initiates cell cycle arrest and cell self-repair.The results showed that there was no significant difference in colony formation ability between 3 c Gy,9 c Gy and 3*3 c Gy groups compared with the control group.The results of CCK-8 assay were consistent with those of colony formation assay.The viability of A549 cells was close to that of the control group at 6 hours and 24 hours after irradiation.Because the previous results showed that DNA damage still existed in the cells of 3 c Gy and 3*3 c Gy groups at 24 hours after irradiation,the cells of the two groups were cultured for 60 generations,and the colony formation ability of the cells of the 20 th,40 th and 60 th generations of each group was detected.The results showed that the cell proliferation ability of the 3 c Gy group and the 3*3 c Gy group was enhanced compared with the control group.The more passages of irradiated cell culture,the more significant the difference in proliferation ability.In addition,when detecting irradiated cells of different generations,the enhancement of cell proliferation in the 3*3 c Gy group was significantly stronger than that in the 3 c Gy group.At the same time,the results of cell viability assay showed that the cell viability of 3 c Gy group and 3*3 c Gy group was higher than that of the control group,and this difference increased with the increase of culture passages.In different generations of cells,the cell viability of the 3*3 c Gy group was more significant than that of the 3 c Gy group.After cell scratch healing width analysis,it was found that the migration ability of cells in the 3 c Gy group and the 3*3 c Gy group gradually increased with the increase of passages,and the scratch healing was completed after 12 hours in our experiment.The results of cell invasion assay showed that the number of transmembrane cells increased significantly with the increase of passages in the 3 c Gy group and the3*3 c Gy group.The number of invasive cells in the 3*3 c Gy group was significantly higher than that in the 3 c Gy group.The above results showed that after low-dose irradiation,the long-term cultured cells had stronger migration ability and invasion ability than the control group,and the 3*3 c Gy group performed more significantly.Then we selected the 40 th and 60 th generations of cells in each dose group to detect their resistance.The cells in each experimental group were treated with 5 μg / ml BLM for 1 hour or placed under 6 Gy X-ray,and the cell viability was determined by CCK-8 method.It was found that the cell viability of the LDCT treatment group was significantly stronger than that of the control group,and the survival rate of the 60 th generation cells was higher than that of the 40 th generation cells,suggesting that low-dose irradiation can improve the tolerance of cancer cells to drugs or irradiation.The subcutaneous tumor model of nude mice showed that the tumor volume of the irradiated tumor-bearing mice in each group decreased,and the tumor volume of the control group decreased significantly after irradiation.The tumor volume of the tumor-bearing mice in the 3 c Gy group and the 3*3 c Gy group began to decrease significantly on the sixth and tenth days after irradiation,respectively.The survival curve of tumor-bearing mice in each group suggested that the survival time of tumor-bearing mice in the irradiation group was shorter than that in the control group,and the survival time of tumor-bearing mice in the 3*3 c Gy group was particularly short,which may be due to tumor load.The results suggest that low dose irradiation also enhances the radiation resistance of tumor cells to varying degrees.The expression of malignant transformation-related proteins E-cadherin,N-cadherin and vimentin in long-term cultured irradiated cells was determined by WB assay.The results showed that the expression of N-cadherin and Vimentin increased in 3 c Gy group and 3*3 c Gy group,while the expression of E-cadherin decreased with the increase of passages.The results showed that low-dose irradiation of 3 c Gy group and 3*3 c Gy group promoted the formation and development of malignant transformation of A549 cells,that is,induced malignant transformation of tumors.At the same time,the expression levels of metabolism-related proteins PKM2 and GLUT1 in the same dose group were significantly increased with the increase of culture passages,suggesting that LDCT can lead to the occurrence and development of malignant transformation(EMT)of lung adenocarcinoma cell A549 from the aspect of glucose metabolism.Four weeks after the establishment of the orthotopic tumor model by tail vein injection in mice,the body weight of nude mice in each group decreased significantly,suggesting that the tumor load increased.Subsequently,PET / CT scan of mice was performed.It was found that the lung signal of mice after irradiation was significantly higher than that of the control group,but no significant metastasis signal was observed.Subsequently,the lung tissues of nude mice in each group were taken for pathological sections.The results of H & E staining showed that the 3*3 c Gy group had the most scattered tumor cell aggregates,followed by the 3 c Gy group,and the control group had the least lesions.The results verified that low-dose radiation can indeed promote the malignant transformation of lung adenocarcinoma cells and metastasis in vivo.Conclusion: Single or cumulative LDCT scanning can lead to the occurrence and development of malignant transformation(EMT)of long-term cultured lung adenocarcinoma cells A549.Despite extremely low doses of irradiation,its long-term effects on cells cannot be ignored.Our experimental results demonstrate some of the effects of LDCT radiation on health status,and provide a reference for the possible effects of undiagnosed pulmonary nodules on tumor development in routine CT examinations. |
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