| BackgroundAcute or chronic inflammation can be induced by ionizing radition, which will damage the immune balance. The important immune regulatory subset is Treg, its change in number, percentage or phenotype will directly influence the equilibrium. For this reason, many studies were recently carried out on radition sensitivity of Treg cells. As the studies on Treg cells were going deeply into, many transcription factors and surface molecules were revealed which were related to Treg cell phenotype and function. However there were differential expression of these proteins in different type of imflammation and infectious tissues. To reveal which protein will be play a role in radiation indueced inflammation, many studies should be carried out.ObjectiveTo analyze the influence of different dose γ-ray irradiation on the number of lymphocytes and their Treg subset, and to explicit the radiation induced changes of apoptosis, proliferation and phenotype of Treg cells. Then to further analyze the relation between apoptosis and differently expressed phenotype factor, and do the expression of microRNAs which target effect on the factor. Then disscuss the mechanism of which the phenotype factor is regulated during its transcription, which will estabilish the theoretical foundation to revover the immune imbalance induced by ionizing radiation.Methods1. Mice were administered with whole body irradiation of γ-rays at different doses, and lymphocytes were separated from thymus and spleen, then the number of total cells were counted and the percentages of CD4+T and CD4+FOXP3+CD25+Treg lymphocytes were analyzed using FCM.2. The lymphocytes in vitro were irradiated by2Gy γ-ray irradiation and the7-AAD combining rate of Treg and Tcon were detected using FCM in9h after exposure. 3. The Annexin-V and Ki-67expression level of Treg and Tcon cells ex vivo were detected as well as the expression level of the transcription factor FOXP3and Helios, the surface molecules CD39and CD103, and apoptosis related proteins Bcl-2and Bax using FCM.4. The microRNAs target effect on CD39were retrieved through different database, the their amplification in Treg cells were analysed using real-time PCR.5. CD4+CD25+Treg cells were sorted and their intracellular ATP concentration were measured using ATP concentration assay kit and microplate reader.Results1. The lymphocyte numbers in thymus and spleen decreased in dose-dependent manner and reached to the minimum at4d after irradiation,(F=118.08,144.01,.P<0.05); Exposure to higher dose(more than1Gy) decreased Treg number time-dependently in thymus, however increased it in spleen; On the contrary, exposure to lower dose(less than0.75Gy) increased Treg number in thymus; Besides the percentage of Treg cells increased dose dependently(in thymus F=5.16,89.44,3.01, P<0.05; in spleen F=52.02,32.13,27.45, P<0.05).2. The survival rate of both Treg and Tcon cells in vitro decreased after irradiation and the latter one decreased more significantly although it was higher in control group.3. The Treg cell apoptosis rate increased significantly and was higher than the one of Tcon cells in4d after irradiation. The apoptosis and proliferation rate of Treg cells were different in different tissues. For proliferation, the percentage of Ki-67+Treg cells in thymus was decreased in1d and reversed in4d, however the trendency was inverse in spleen.4. The percentage of FOXP3+Treg cells increased after irradiation and the MFI of FOXP3in Treg cell was decreased in thymus and increase in spleen. Both striking changes were happened in10d after irradiation. Besides the expression level of Helios kept relatively stable.5. The CD39and CD103of Treg cells were differently expressed after irradiation. The percentage of CD39+Treg cells increased and CD39expression level of Treg cell was upregulated in thymus but inverse in spleen. The changes of the percentage of CD103+Treg cells were complicated than CD39, it was firtly increased then reverse in spleen, however there was a transient increase in thymus in4d. and CD103expression level of Treg cell was down regulated in thymus but inverse in spleen.6. It was Bax that its MFI significantly increased but the Bcl-2in thymus and it was reverse in spleen that the MFI of Bcl-2increased, which resulted in the ratio of Bax/Bcl-2increase in thymus and inverse in spleen.7. Six microRNAs were found which target effect on CD39, and the expression changing trendency of miR-31was similar with the one of CD39.Conclusion1. The cell number changes of lymphocytes and their Treg subset in dose dependent manner, and there are different time-response between lymphocytes and their Treg subsets.2. Treg cells in vitro were resistant to irradiation, however the apoptosis rate of the ex vivo ones increased significantly after irradiation. The proliferation rate of Treg cells induced by radiation changes in time and tissue dependent manner, which revealed that the survival of Treg cells are significantly influenced by the microenvironment.3. FOXP3of Treg cells expressed differently after irradiation as well as the phenotype factors CD39and CD103. The CD39is related to the radiation induced apoptosis and CD103helps Treg cells immigration.4. Bax and Bcl-2respectively take part in the apoptosis program of Treg cells from thymus and spleen. And CD39may play a role in the program.5. The expression of CD39can be regulated by genes in which miR-31may play a negative role. |
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