| Background and objective:Radiotherapy is an important treatment for head and neck cancer.However,complications occur in more than 60% of surgical patients after radiotherapy.Major clinical sequelae include skin atrophy,soft tissue fibrosis,peeling,epithelial ulcer,fistula formation and rupture of great vessels.Perioperative and postoperative wound healing is often slowed down,and the reconstruction process after skin radiotherapy becomes very difficult.In skin wound healing,skin fibroblasts play a key role in the whole process of wound healing.As a tumor suppressor gene P53 gene,plays an important role in cell apoptosis,the aim of this study is to set up a radiation model based on mice and human skin fibroblasts.This model should follow the need of clinical practice.Another aim of this study is to find the relationship between wound refractory healing and P53 apoptosis pathway and provide a new way of thinking for clinical practice.Methods:1.Establishment of mouse and human skin fibroblast models that can be passaged after radiation.Cultured skin fibroblasts were digested by trypsin and then suspended in 15 ml Centrifuge tube.10 Gy radiation was given at the radiation source,and the cultured skin fibroblasts could be passed on normally after radiation.The same radiated cell lines in which human skin fibroblasts could be passed on were constructed.2.The ability of proliferation,apoptosis and migration of radiated passable cell lines in mice and humans was tested to determine the difference between radiated and non-radiated cells.3.Complete transcriptome sequencing was performed on fibroblast cell lines that had been passaged for a long time,and changes in the P53 pathway were observed to verify whether the original experimental inference was correct.4.The P53 pathway was detected at the protein level to observe the changes in the protein level and the similarities and differences with the transcription level.5.Blocking the P53 pathway with P53 inhibitors,and observed the proliferation of related radiated cells after blocking the P53 apoptosis pathway.Results:1?CCk-8 cell proliferation experiment results showed that the proliferation of the first generation of mouse fibroblasts after radiation was significantly slower than that of the non-radiated group,and there were statistically significant differences between the control group on the third day and the fifth day(P<0.05)Microscopic photographs,according to the results of the first generation of cells under g-ray,no obvious change in the process between the first generation.When the cells were subcultured,the cell appearance changed gradually.Immunofluorescence staining results also showed that cell proliferation was reduced after radiation,and proliferation-related antibody Ki67 was significantly reduced in the radiation group(figure 1-4).On the basis of the mouse skin cell radiation model,the human skin fibroblast model was constructed.The results of cck-8 were similar to mouse fibroblast radiation,but there was no significant statistical difference on the third day(P>0.05),and the difference was significant on the fifth day(P<0.01).Compared with the results of mouse fibroblasts,the proliferation curve of human skin fibroblasts showed a plateau period after the same dose of gamma ray radiation.The cell appearance changed from short spindle to long oval.The cytoplasm was loose and the cell membrane of some cells was clear and bright,and a few nuclei became dark.2?With FITC and PI double stained,we found that early apoptosis and late apoptosis cells increased after radiation,.The number of apoptosis cells of the third generation compared with the first generation,have no statistical difference(P>0.05).Results of human skin fibroblasts and the results of mouse cells were similar,the cell number of early apoptosis and late apoptosis in radiated group was increased,the total number of apoptosis cells was statistically difference compared with control group(P < 0.05).It was also found that total number of apoptosis cells in the third generation still had significant statistical difference compared with the third generation of the control group(P < 0.05).But different from mice fibroblast,the apoptosis cells of the third generation of human skin fibroblast are equivalent to the first generation.3?Compared with the control group,the first generation of radiated mouse skin fibroblasts showed no significant reduction in the migration rate(P>0.05),and the third generation of radiated mouse skin fibroblasts showed slower migration rate compared with the control group(P<0.05).The migration rate of radiated mouse skin fibroblasts decreased with the increase of cell subculture.After repeating the above experiment in human skin fibroblasts,the results were consistent with those of mouse skin fibroblasts,that is,cell migration slowed down with the increase of subculture,and there was a statistical difference between the third generation of radiated cells and the control group(P<0.05).4?H2a?-labeled nuclei were found in the third generation of radiated mouse skin fibroblasts,and the number of H2a?-stained positive cells in the radiated group was significantly higher than that in the control group.5?After radiation,the cell cycle of mouse skin fibroblasts was significantly changed,the G1 phase was shortened in the early phase of DNA synthesis,while the G2 phase was prolonged in the late phase of DNA synthesis,and there was no significant change in the S phase and M phase.6?The RNA-seq Results showed that the P53 pathway raised significantly.The downstream target gene P21 levels raised in the first generation and the third generation,P53 pathway was activated after radiation.The result of human skin fibroblasts is similar to the findings of mouse.The P53 pathway in whole transcriptome sequencing showed the same result,the amount of P53 pathway gene expression was lower compared with the mouse cells,the protein experiments testing found that after the first generation and the third generation of P21 protein content were increased in radiated cells,prompt P53 in the activated state.7?After 3 days(72h)of the third generation of mouse cells after inhibitor action,it was found that the number of cells in the inhibitor group increased,which was significantly different from that in the DMSO group.Conclusions:Our experiment established a new model for the radiation injury in mice and human fibroblasts.On the basis of this model further study found that the slow of wound healing is caused by some special factors.The existing cellular proliferation continues to slow down for a long time after radiation,the phenomenon of apoptosis continue to increase,the migration rate is affected with subculture.The RNA-Seq result showed abnormal activation of P53 apoptosis pathways in the radiated cells.When the P 53 apoptosis pathway was blocked by P53 inhibitor,the cell proliferation could partially be restored.This provides a new idea for wound healing with radiotherapy. |
PDF Full Text Request