| Objective: By investigating the changes of leukocytes,T lymphocytes(TLC)and subpopulations of it(including CD3+,CD4+,CD8+,CD4+/CD8+,Treg,etc.),and dendritic cells(DC),macrophages(M?)and their surface molecules in lung tissues and lung drainage lymph nodes(LDLN)early after ionizing radiation.Explore the changes in immune cells and antigen presenting processes in lung early after ?-ray irradiation of the mouse chest(3h to 14d).The findings may provide the basis for mechanisms of radioactive lung injury and early intervention measures.Methods: 1.Animal experiments:(1)Irradiation: to establish a model of irradiation to the chest of mice: 280 C57BL/6 male mice(6-8 weeks,20±2g)were randomly divided into 14 groups(7 groups each of control and irradiation),20 mice per group.(2)Extraction and detection of primary cells: mice were executed at 3h,12 h,1d,2d,3d,7d and 14 d after irradiation,lungs and drainage lymph nodes were removed,cut,digested and isolated with collagenase,and filtered through 40?um cell sieve to obtain singlecell suspensions of lungs and drainage lymph nodes,and the cells were labeled by antibodies,then the changes in cell surface markers were detected by flow cytometry at different times after irradiation,mainly to observe the changes in DCs and co-stimulatory factors on the cell surface,T cell subpopulations was detected too.2.In vitro co-culture experiments:(1)Mice spleen cells were co-cultured with the irradiated mice bronchial epithelial cells(MLE-12),and antibodies were labeled at 3h,6h,and 24 h.Then the changes in T cells and their surface co-stimulatory molecules were detected by flow cytometry.(2)Dendritic cells(DCs)differentiated from mouse bone marrow were co-cultured with irradiated MLE-12 and labeled with antibodies at 6h,24 h and 48 h respectively,then the changes in DC cell surface markers(CD11C,CD80,CD86,MHC?,MHC?)were detected at different time points.Results: 1.The number of white blood cells in the lung tissue of mice in the irradiation groups was significantly reduced(P <0.05);CD3+ T cells decreased early(3h ~ 2d)after irradiation,and the difference from the control group was statistically significant(P <0.05);Both CD4+ T and CD8+ T cells showed volatility changes,but there was a significant time difference,CD4+ T cells were significantly reduced early(3h and 12h)after irradiation(P <0.05),and volatility recovered after 1d to the level of the control group;CD8+ T cells did not change significantly at the early stage(3h and 12h)after irradiation,then decreased significantly at the first and third day(P<0.05),and significantly increased at the 7th(P<0.05);corresponding to this,the ratio of CD4+/CD8+ decreased significantly(P<0.05)in the early period(within 1d)and late period(after 7 days),and volatility increased during the period(P<0.05);Treg was significantly higher than the control group,the difference was statistically significant(P<0.05);The proportion of DCs was significantly higher than that of the control group at each time point after irradiation(P<0.05);The expression of CD80 in the lung tissue of mice in the irradiation groups increased significantly from 3h to 3d(P<0.05).After 7 days,the expression of CD80 returned to normal level;the expression of CD86 on DC surface was higher than that of control group from 12 h to 7th day(P<0.05);the expression of MHCI on DC surface was High expression(P<0.05)in the period(within 3days);MHCII expression on DC surface was lower than that in the control group from 3h to 336h(P<0.05);macrophages(M?)did not change significantly after irradiation(P>0.05);The expression of CD80 on the surface of M? increased significantly on the second day(P<0.05),and returned to normal levels on the third;the expression of CD86 on the surface of M? increased significantly on the third(P<0.05),and the expression of CD86 returned to normal levels after that(P>0.05);MHCII expression on the M? surface decreased significantly from second to 7th(P<0.05),and returned to normal level after 14 th.2.Compared with the control group,the leukocytes in the drainage lymph nodes of the irradiation group were significantly reduced(P<0.05),and recovered after 14 days,but still lower than the control group(P<0.05);3h to the 7th day after irradiation,CD3+ T cells were significantly increased(P<0.05),and recovered after 14 days,and were at same level with control group(P>0.05);after irradiation,CD4+ T cells showed volatile changes,but there was a significant time difference,significantly decreased in the early and late period(3h and 12h)(P<0.05),higher than the control level from the first day to 7th day(P <0.05),and recovered after 14 days(P>0.05);CD8+ T cells did not change significantly in the early 3h after irradiation(P>0.05),decreased significantly from 12 h to the 7th day(P <0.05),recovered after 14 days,and remained the same as the control group(P>0.05).Correspondingly,the CD4+/CD8+ ratio did not change in the early(within 1d)and late(after 14 days)after irradiation(P>0.05),and was significantly higher than that in the control group from 1d to 7d(P <0.05);the Treg ratio was all higher then the control group from 24 h to 14 d.The number of DC is above the control group,showing fluctuations.There is no difference between irradiation groups and control groups at 3h and 7d(P> 0.05),but irradiation group is higher control group at 12 h to the third day and the 14 th day(P <0.05);the expression of CD80 and CD86 on the DC surface of the irradiated group was higher than that of the control group at 12 h to 14 th day(P <0.05);the expression of MHCI on the DC surface was high in the early(12h)and the 7th day,the 14 th day(P<0.05);The proportion of MHCII on the DC surface was lower than that in the control group from 12 h to the 7th day(P <0.05).3.Spleen cells were co-cultured with MLE-12 in vitro,and the expression of CD28 on the surface of CD3+,CD4+,and CD8+ T lymphocytes was higher than that of the control group at 6h and 24h(P<0.05);DC and MLE-12 were co-cultured in vitro culture,DC was highly expressed from 6h to 48 h after irradiation,DC surface marker CD80 / 86,and MHCI/II were highly expressed at 6h after irradiation(P<0.05).Conclusion: In mice irradiated with chest,early T cell responses in lung tissue showed different changes,possibly related to radiation-induced immune cell damage and immune responses generated by organismal stress.DC cells increased and DC surface co-stimulatory factors CD80,CD86 and MHCI were persistently expressed,indicating radiation-activated lung DC antigen presenting capacity.The in vitro co-cultivation experiment preliminarily explored the mechanism of irradiation-induced antigen presentation.It is speculated that irradiation activates DC antigen presentation by inducing lung epithelial cell damage to activate T cells. |
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