Anti-tumor Effect And Mechanism Of GSK-3β Knockout In Thymic Epithelial Cells In Aging Mice

Background:Thymus is one of the earliest aging organs in human body.thymic epithelial cells（TECs）,as an important component of the thymus microenvironment,are the necessary nurturing cells for the body to produce T cells with normal function.With the increase of age,the number and function of TECs decrease,the production of normal T cells decreases and the immune function declines.Anti-tumor ability becomes weak.How to slow down this process and enhance anti-tumor ability has become a research hotspot.Purpose:Explore TECs-specific knockout glycogen synthase kinase-3βconstructed by Cre-Loxp technology.The number changes of thymus epithelial cell subsets and related functional molecules,the number changes of thymus lymphocyte subsets,and the number changes of lymphocyte subsets in spleen and peripheral blood of mice in aging mice with GSK-3βgene.To observe the anti-tumor ability of GSK-3βgene knockout mice with TECs specificity.To clarify the difference between its anti-tumor ability and that of Wild type（WT）mice and explore its mechanism,so as to provide ideas and experimental basis for enhancing anti-tumor immunity of the body by delaying the decline of thymus function clinically.Methods:GSK-3β^floxp（GSK-3β^f）mice with C57BL/6J background were paired with Fox N1^cremice,and the offspring were genotyped by DNA gel electrophoresis.The mice with different genotypes were grouped.GSK-3β^f/fFox N1^crewere Conditional knockout（cKO）mice group,and other WT mice in the same colony were control group.The thymus of cKO mice and WT mice were stained with Keratin 5（K5）,Keratin8（K8）,GSK-3βand Ki67 fluorescent antibodies,respectively.The structure of thymus tissue and the expressions of K5,K8,GSK-3βand Ki67 proteins were observed by Confocal laser scanning microscope（Confocal）.Therefore,the difference between cKO mice and WT mice was further determined by the expression location of GSK-3βprotein in fluorescence superposition map,and then Flow Cytometry was used to detect the difference between cKO mice and WT mice.FCM were used to detect the changes of TECs,thymus cells and their subsets,lymphocytes and their subsets in spleen and peripheral blood,and related cytokines in mice aged 6 weeks,6 months,12 m,and 24 m.Mice aged 12 m were subcutaneously loaded with pancreatic tumor PAN02 cells to observe the changes in body weight and tumor volume.The mice were sacrificed at the end of the experiment,and the changes of invasive T cells and related cytokines in tumor tissue were detected by FCM.Spleen lymphocytes of mice with pancreatic tumor were co-cultured with Pan02 tumor cells and B16F10 melanoma cells,respectively.After 48 hours（hours,h）,the proportion of lymphocytes and their subsets,secretion of cytokines and the ability to kill tumors were detected.Results:DNA gel electrophoresis can clearly distinguish the genotypes of mice.Confocal observed that GSK-3βwas not expressed on K5⁺and K8⁺cells（K5 and K8 were expressed on cortical TECs and medulla TECs,respectively）,namely,the TECs-specific knockout mice were successfully constructed.In addition,Ki67 expression of TECs was higher in knockout mice,and thymus volume increased significantly without significant fat infiltration.The results of FCM showed that the number of TECs and early thymic progenitor cells（TEPC）increased in cKO mice,and the expression of TECs Aire and CD80 was enhanced.The number of thymus cells and their subgroups increased significantly.The number of lymphocyte subsets in spleen and peripheral blood of cKO mice increased significantly compared with WT mice.The anti-tumor cytokines IL-2,IFN-γand TNF-αincreased,while the pro-tumor cytokines IL-10 and TGF-βdecreased.Compared with WT mice at the same developmental stage,12 m old cKO mice exhibited slower growth of pancreatic tumors.FCM was used to detect the number of lymphocytes in the tumor microenvironment of cKO and WT mice.It was found that the proportion of invasive T cells in tumor tissue increased,the number of anti-tumor cytokines secreted by various immune cells increased,and the expression of lymphocytes surface activating molecules increased.Spleen lymphocytes of cKO mice and WT mice after being loaded with pancreatic tumor were isolated in vitro and co-cultured with Pan02 tumor cells and B16F10 melanoma cells.It was found that in the co-culture system of cKO mice,the number of various lymphocyte subgroups increased,the secretion of anti-tumor cytokines increased,and the secretion of pro-tumor cytokines decreased.CD8⁺T lymphocytes and Granzyme B（Gzb）secretion increased.Conclusion:Compared with WT mice,cKO mice have more TECs in the aging process,more mature T cells can be produced,and have stronger immune function.The anti-tumor ability of TECS-specific knockout GSK-3βgene mice at the aging stage was significantly stronger than that of wild-type mice at the same developmental stage.This provides a corresponding idea for delaying thymus senescence in clinical practice to achieve the purpose of anti-tumor.