| Glioma is a kind of malignant tumor that originates from central glial cells,and it is the most common intracranial primary tumor.At present,surgery combined with postoperative concomitant chemoradiotherapy is the standard protocol for glioma treatment,which can improve the cure rate,median survival time,progression-free survival and overall survival of glioma.In recent years,with the further study of radiobiology and the development of radiotherapy technology,radiotherapy has become one of the important methods of glioma therapy.Based on the difference of histological type and histological grading of glioma,the effect of radiotherapy is different.For example,the lethal effect of radiotherapy on different histological grading of glioma is significant different,especially grade III-IV astrocytomas,which have poor sensitivity to radiotherapy alone accompanying poor efficacy,usually relapsing at the same or higher grading forms.The Studies have shown that there is a hypoxia microenvironment in glioma,and hypoxic microenvironment is an important factor leading to resistance of glioma to radiotherapy and chemotherapeutic drugs.Hyperbaric oxygen（HBO）is an effective method to change the hypoxic state of tumor tissue,which can increase oxygen concentration in blood and increase the diffusion radius of oxygen in tissue,Thus improving the hypoxia microenvironment of tumor tissue.Therefore,we hypothesize whether hyperbaric oxygen can improve the hypoxic state of tumor tissue to improve the radiosensitivity of glioma,and then improve the prognosis of glioma patients,which is verified by this experiment.Objectives:To investigate the value of HBO in improving the radiosensitivity of glioma,through observing the effects of HBO combined with X-ray irradiation on cell proliferation activity,cell cycle distribution and apoptosis of human glioma U251 cells.Methods:Human glioma U251 cell line after subcultured were randomly divided into control group,HBO group,radiation（0,2,4,6,8Gy）group,HBO+radiation（0,2,4,6,8Gy）group,observing the differences in cell morphology,cell proliferation activity,cell cycle distribution and apoptosis between groups:（1）Observing Cell adherence and cell status in different treatment groups;（2）Observing cell survival situation with clone formation experiment,then the survival curve of U251 glioma cells was fitted by linear quadratic equation,and applying click multi-target model to calculate radiobiological parameters（D0,Dq,SER）for Evaluating the effect of HBO on glioma radiosensitivity;（3）Using CCK-8 experiment to detect the effect of HBO and different doses of X-ray irradiation on glioma U251 cells proliferation;（4）Applying FCM experiment to analyze the effects of HBO combined with X-ray irradiation on cell cycle distribution and apoptosis of glioma U251 cells.Results:（1）After 24h,48h and 72h of cell culture,the cell counts in HBO+X-ray irradiation group[（1.04±0.13）×106,（0.36±0.06）×106,（0.08±0.04）×106]were significantly lower than those of the X-ray irradiation alone groups[（1.94±0.09）×106,（0.79±0.09）×106,（0.43±0.10）×106]（P<0.05）.（2）Survival fraction（SF）of U251 glioma cells in X-irradiation（0,2,4,6,8Gy）group were1.000±0.000,0.660±0.021,0.424±0.040,0.301±0.047 and 0.075±0.010,respectively;SF of U251 glioma cells in HBO+X-irradiation（0,2,4,6,8Gy）groups were 0.750±0.023,0.319±0.025,0.235±0.020,0.109±0.025 and 0.019±0.015,respectively.There were significant difference in SF between HBO combined with X-irradiation group and X-irradiation group（P<0.05）.D0 and SF2 in X-irradiation group were 4.01 and 2.64,respectively;that of HBO+X-irradiation group were 0.660 and 0.319,respectively.SER（D0）and SER（Dq）in HBO+X-irradiation group were 1.52 and 2.02,respectively,which indicates that HBO can significantly enhance the sensitivity of glioma U251 cells to radiation.（3）The proliferation inhibition rates of U251 glioma cells in X-irradiation（2,4,6,8Gy）group were 0.1026,0.1530,0.2157 and 0.2327,respectively;Those in HBO+X-irradiation（2,4,6,8Gy）group were 0.2039,0.2622,0.3143 and 0.3360,respectively.There was a significant difference in the proliferation inhibition rates of U251 glioma cells between two groups（P<0.05）.（4）The proportion of G2/M phase cells in HBO group（22.36±0.91）and HBO+X-ray irradiation group（26.70±2.46）were significantly higher than those in the corresponding control group（11.56±2.01）（P<0.05）.However,there was no significant difference in the proportion of G2/M phase cells between X-irradiation group and the control group（P>0.05）;The proportion of G2/M phase cells in HBO+X-ray irradiation group were significantly higher than those in the X-ray irradiation group（P<0.05）.However,there was no significant difference in the proportion of G2/M phase cells between HBO+X-irradiation group and the HBO group（P>0.05）.（5）The apoptosis rates of U251 cells in X-irradiation group,HBO group,HBO+X-irradiation group and control group were 11.62,11.22,30.99 and 5.59,respectively.The apoptosis rate of U251 glioma cells in HBO+X-irradiation group were significantly higher than those in X-irradiation group,HBO group and control group.Conclusion:（1）Hyperbaric oxygen can reduce the activity of glioma U251 cells and inhibit its proliferation.Hyperbaric oxygen combined with X-irradiation can increase the inhibition rate of radiation on glioma U251 cell proliferation,as well as increasing the apoptosis rate of glioma U251 cell.Therefore,hyperbaric oxygen plays an important role in radiosensitivity of glioma.（2）Hyperbaric oxygen can induce G2/M phase arrest of glioma U251 cells,and G2/M phase cells are highly sensitive to radiation,which elucidates the preliminary mechanism of hyperbaric oxygen increasing radiosensitivity. |
