| Objective CBX4(chromobox 4)has been shown to be critical for the maintenance of thymic function and peripheral T cell numbers in embryonic and neonatal mice.CBX4-specific deletion in thymic epithelial cells(TECs)of embryonic mice leads to congenital dysplasia of the thymus,but its effect on TECs function in adult mice is unclear.This study successfully constructed an inducible knockout CBX4 adult mouse model to explore the regulation of CBX4 on the function of TECs and thymus development in adult mice,and its effect on peripheral T lymphocytes.Methods First,Western Blot technology was used to detect the protein expression level of CBX4 in various tissues and organs of adult mice,and secondly,HE staining and IHC were used to identify specific expression sites of CBX4 in their tissues and organs.Secondly,in the Cre-ERT mouse model,the experimental group used estrogen-Tamoxifien to induce systemic knockout of CBX4,and mice of the same genotype were injected with an equal volume of corn oil as the control group.After the DNA and protein levels were successfully knocked out,flow cytometry was used to detect the development of the thymus and the proportion and activation of peripheral T cells in the two groups of mice.Then,HE staining was used to observe the effect of CBX4 knockout on the structure of adult mouse thymus.Finally,immunofluorescence was used to detect cytokertain 5(K5)and cytokeratin 8(K8),Aire and CD205 were differentially expressed in thymic epithelial cells of the two groups of mice.Results 1.Western Blot results showed that CBX4 protein levels were higher in the spleen,lung,brain,lymph nodes and thymus of adult mice.HE staining and IHC showed that CBX4 was highly expressed in the red marrow of adult mouse spleen,lung alveoli,pons,brain microglia,lymph node medulla,thymus cortical epithelial cells(c TECs)and medullary epithelial cells(m TECs).2.The results of live cell count showed that the total number of thymocytes in the CBX4 knockout group was significantly lower than that in the control group(P<0.001),and there was no difference in the total number of peripheral spleen cells and lymph node cells compared with the control group.3.The results of flow cytometry showed that compared with the control group,the number and proportion of double-positive thymocytes in the CBX4 knockout group were significantly decreased(P<0.001);the proportion of double-negative thymocytes was significantly increased(P<0.01);The number of CD4 single-positive thymocytes was significantly decreased(P<0.05);the number of CD8 single-positive thymocytes was significantly decreased(P<0.05).Based on the expression of CD44 and CD25,in the further grouping of doublenegative thymocytes,the number and proportion of DN1 thymocytes were significantly increased(P<0.05);the number and proportion of DN4 thymocytes were significantly decreased(P<0.05).However,there was no significant difference in the number and activation ratio of peripheral T cells compared with the control group.4.The results of HE staining showed that the thymic epithelial structure of CBX4 knockout adult mice was significantly atrophied,and the cortex and medulla structure were greatly reduced.5.The immunofluorescence results showed that compared with the control group,the expression of K5 in thymic epithelial cells of CBX4 knockout adult mice was significantly reduced;the expression of K8 was significantly reduced;the expression of Aire was significantly reduced;the expression of CD205 was significantly reduced.Conclusions Our research shows that CBX4 is essential for the maintenance of the function and structure of adult mouse thymic epithelial cells(TECs).The loss of CBX4 can cause damage to the proliferation of cortical epithelial cells(c TECs)and medullary epithelial cells(m TECs),which in turn affects the thymus Positive selection and negative selection of cells lead to severe dysplasia of the thymus.However,the differentiation and activation of T cells in peripheral immune organs has no effect. |
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