| Objective To observe the trend of CD4~+CD25~+regulatory T lymphocytes(Treg),CD8~+effector T cells(Teff)and inhibitory cytokine COX-2 in patients with esophageal squamous cell carcinoma(ESCC)before,during and after radiotherapy.The effects of radiotherapy on the immune microenvironment including Treg,Teff and inhibitory cytokines were verified in the ESCC mouse xenograft model,and the effect of Celecoxib,a cyclooxygenase inhibitor,on increasing radiotherapy sensitivity by regulating Treg to affect the immune microenvironment was further studied and the possible mechanism was explored.Methods In the first part,the peripheral venous blood of 30 ESCC patients receiving radiotherapy was collected,then Flow CytoMetry(FCM)and Enzyme linked immunosorbent Assay(ELISA)were used to determine the levels of Treg cells,Teff cells and COX-2 in the blood of these patients before and after radiotherapy.In the second part,an ESCC animal model was established by injecting ECA109 cell line into the right forearm of BALB/c mice,24 mice were randomly divided into four groups(group A,group B,group C,and group D)after pathologically confirmed tumor success,and were given corresponding therapeutic intervention(blank control,celecoxib therapy,radiotherapy,and celecoxib combined with radiotherapy).From the 10th day after inoculation of the transplanted tumor,the tumor growth size and body weight of mice were recorded regularly every 3 days,and the growth rate curve of the transplanted tumor was plotted.Meanwhile,group B and D were given celecoxib intragastric administration,while group A and C were given the same volume of sterile normal saline(0.9%sodium chloride aqueous solution)intragastric administration,which lasted until the end of the experiment.On day 20,group C and group D were treated with local 12Gy electron line irradiation.On day 30,the mice were killed and the whole blood was collected from eyeballs and centrifuged for serum.FCM and ELISA were used to detect and compare the contents and differences of Treg cells,Teff cells,immunosuppressor cytokines(TGF-β,COX-2,IL-10)in the peripheral blood of tumor-bearing mice.Results 1.With the increase of days,the proportion of Treg cells in peripheral blood of ESCC patients during and after radiotherapy increased from the baseline value(11.47±2.74)%,and the proportion of Teff cells decreased from the baseline value(22.03±6.72)%,(all P<0.001).2.The expression level of COX-2 in ESCC patients increased during and after radiotherapy,which was different from the baseline value(all P<0.001).3.In ESCC mice transplanted tumor model,the difference between the radiotherapy group and the untreated group was obvious,the proportion of Treg in peripheral blood of the former showed an upward trend(15.42±0.62)%VS(10.05±0.48)%,P<0.01,while Teff cells showed a downward trend(4.25±0.25)%VS(5.24±0.46)%,p<0.001,and the expression of COX-2 was gradually increased(52.70±16.11 ng/ml VS34.58±3.35ng/ml,p<0.01).There were also obvious difference between the celecoxib group and the untreated group,the proportion of Treg in peripheral blood of the former showed a downward trend(8.52±0.47)%VS(10.05±0.48)%,p<0.01;while Teff cells showed a upward trend(8.07±1.36)%VS(5.24±0.46)%,p<0.01;and the expression of immunosuppressant factor was decreased(TGF-β,0.14±0.08 ng/ml VS 0.52±0.33ng/ml,p<0.05;COX-2,25.91±5.73 ng/ml VS 34.58±3.35 ng/ml,p<0.05;IL-10,329.32±73.62 ng/L VS 430.25±43.63 ng/L,p<0.05).The proportion of Treg in the combined treatment group were less than radiotherapy alone group(11.09±0.35)%VS(15.42±0.62)%,p<0.01;the proportion of Teff in the combined treatment group were higher than radiotherapy alone group(6.65±0.65)%VS(4.25±0.25)%,p<0.01;and the expression of immunosuppressant factor in the combined treatment group were all lower than radiotherapy alone group.(TGF-β,0.51±0.37 ng/ml VS 1.63±0.25 ng/ml,P<0.001;COX-2,26.10±5.81 ng/ml VS 52.70±16.11 ng/ml,P<0.01;IL-10,406.22±63.53 ng/L VS 501.34±56.35 ng/L,P≤0.05).4.Compared with radiotherapy alone,tumor size and growth rate curves were significantly slower and slower in the combined treatment group,and tumor weight was significantly reduced(0.06±0.01g VS 0.17±0.08g,P<0.05).Conclusion 1.Radiotherapy can cause changes in the immune microenvironment,mainly resulting in the increase of COX-2 expression and the increase of Treg cell number.2.Celecoxib can reduce Treg cells,TGF-β,IL-10,COX-2 and other immunosuppressive factors in peripheral blood of ESCC tumor-bearing mice.3.Celecoxib combined with radiotherapy inhibited the growth of transplanted tumor in mice.4.Celecoxib exerts radiotherapy sensitization by inhibiting Treg cells and down-regulating inhibitory cytokines. |
