| Background Immunosenescence refers to the structural destruction of immune organs and immune response dysfunction,resulting in congenital and adaptive immune damage,leading to changes in the immune function of the elderly,accompanied by a systemic inflammatory state characterized by chronic inflammation,and the occurrence and development of infection,autoimmune diseases and tumors are closely related.At present,the exact mechanism of immunosenescence has not yet been fully clarified.The destruction of the structure and function of primary and secondary lymphatic organs can lead to the decline of immune function,so the aging changes of immune organs may be related to the occurrence of immunosenescence.The thymus and spleen are the body's largest central and peripheral immune organs.As age increases,the structure and function change,the destruction of the normal microenvironment and the decrease in the number of T lymphocytes cause the decline of the body's immune function and affect the homeostasis of peripheral immunity.It may promote age-related inflammation of the body and the occurrence of immunosenescence.The changes in the structure and function of the thymus and spleen caused by aging may be related to immunosenescence.Objective The molecular changes of thymus and spleen with aging and the mechanism are not yet clear.Based on the integrated transcriptome and proteomics methods,this experiment reveals the age-related molecular changes of mouse thymus and spleen tissues of different age groups at the transcriptome and proteome levels,and explores the effects of age-related changes in thymus and spleen on immune aging Potential role in the medium,providing possible candidate targets for the diagnosis and treatment of related diseases caused by immunosenescence.Methods In this experiment,the histological changes of the immune organs of the thymus and spleen of mice of different age groups(6-weeks,6-months,and 20-months)were studied by HE staining,immunohistochemistry,and ultramicroscopic observation by transmission electron microscopy.Subsequently,through the integration of RNA-seq transcriptomics methods and tandem mass spectrometry(TMT)relative quantitative proteomics technology,the difference in expression of mouse thymus and spleen tissues of different age groups at the transcriptome and proteome levels was studied,combined with biological information The method analyzes the enriched pathways,screens out the differential genes and proteins related to aging,and further verifies the screened differential genes and proteins.Results1.Changes in the weight and index of immune organs in mice with aging The weight of mice increases with age.The weight of the mouse thymus decreased with age.The weight of the mouse thymus in the20-month-old group and the 6-month-old group was significantly lower than that in the 6-week-old group,and it was statistically significant.The weight of the spleen of mice did not change significantly with age.The weight of the spleen of mice in the 20-month and 6-month-old groups was higher than that in the 6-week-old group,and there was no statistical difference.Both the thymus index and spleen index of mice decreased significantly with age.2.Histological changes of immune organs(1)Thymus HE staining: In the 6-week-old group,the thymus cortex and medulla of mice are clearly demarcated,the cortex is darkly stained,and the lymphocytes are densely stained;the medulla is slightly stained,the structure is slightly looser,the number of lymphocytes is less than that of the cortex,and there are a lot of TECs.In 6-month-old mice,the cortical components of the thymus tissue became thinner,the thymocyte space in the cortical medulla increased,and the skin/medullary ratio was significantly reduced;the number of TECs in the medulla was less.Compared with the 6-week and 6-month-old mice,the thymus tissue of the20-month-old group was significantly reduced and the area was significantly reduced.The surrounding connective tissue was surrounded and replaced,and the cortical and medulla boundary was unclear;the numbers of thymocytes and TECs were less,and the thymus The cells are deeply stained and irregular,and the number of fibroblasts increases. Immunohistochemistry: With the increase of age,the number of CD3-positive T lymphocytes and Ep CAM-positive TECs in mouse thymus skin and medulla decreased significantly.Cortical TECs in the20-month-old group were significantly lower than those in the 6-week-old and 6-month-old groups,and the medullary TECs in the 20-month-old and6-month-old groups were significantly reduced compared with the6-week-old group;the cortical lymphocytes in the 20-month and6-month-old groups were 6 The week-old group decreased significantly.The medullary lymphocytes of the 20-month-old group were significantly reduced compared with the 6-week-old and 6-month-old groups;the macrophages of the 20-month-old and 6-month-old groups were significantly increased compared with the 6-week-old group. Ultrastructure: As the mouse thymus grows with age,the number of cells decreases,the structure is loose,the lymphocyte nucleus tends to be irregular,and the nucleus-to-cytoplasm ratio is small;necrotic thymus epithelial cells can be seen.In the 20-month-old group,there was an increase in apoptosis,and mitochondria of epithelial cells showed vacuolation.(2)Spleen HE staining: In the 6-week-old group,the red and white pulp and marginal structure of the spleen are clear,the spleen corpuscles are regular,the lymphoid follicles are deeply stained,and the lymphocytes are dense.In the 6-month-old group,the boundary between the red and white pulp of the spleen was blurred,the marginal area was significantly wider than that in the 6-week-old group,the germinal center area was enlarged,and the lymphocytes were slightly looser than that in the 6-week-old group.In the20-month-old group,the boundary of the cortex and medulla was blurred,the spleen corpuscle structure was irregular,the white pulp was atrophy and the area was reduced,the red pulp area was correspondingly increased,and the red/white pulp ratio was significantly higher;the marginal area was similar to that in the 6-month-old group.The 6-week-old group was significantly enlarged,the germinal center area increased,and fibroblasts and fibrous connective tissue proliferated in the parenchyma. Immunohistochemistry: With the increase of age,the number of lymphocytes in the white pulp of mouse spleen decreased significantly,the number of B lymphocytes in the white pulp marginal zone and germinal center increased,and the number of macrophages in the marginal zone increased significantly with aging. Ultrastructure: The lymphocytes in the spleen of 6-week-old mice are denser,the nucleus is round,the nuclear to cytoplasm ratio is large,the heterochromatin is more,and the cytoplasmic mitochondria,endoplasmic reticulum,and powerful ribosomes are more abundant;locally scattered Neutrophils,plasma cells,macrophages,etc.The lymphocytes in the spleen of 6-month-old mice have slightly irregular nuclei,heterochromatin gathers at the edge,and mitochondrial vacuolation can be seen in part of the cytoplasm;apoptotic cells increase.The lymphocytes in the spleen of20-month-old mice are loose,the number is obviously reduced,the nucleus is irregular,the heterochromatin is gathered,and the apoptotic cells are obviously increased.3.ELISA test The cytokine results showed that the cytokines IFN-?,IL-10,TNF-? and IL-6 in the serum of mice showed a tendency to increase with aging.IL-10,TNF-? and IL-The level of 6 was higher than that of the 6-week-old group and the 6-month-old group,and the IFN-?level in the 20-month-old and 6-month-old group was higher than that of the 6-week-old group,and there was no statistically significant difference.4.Analysis of Thymus Transcriptome and Proteomics Results RNA-Seq method screened out 524 thymus age-related differentially expressed genes,which are mainly involved in signal transduction processes(PI3K-Akt signaling pathway,Rap1 signaling pathway,Ras signaling pathway),signaling molecules and interaction processes(ECM-receptor interaction Role),immune system(B cell receptor signaling pathway,complement and coagulation cascade,chemokine signaling pathway)and other signaling pathways;TMT technology determines 113 differentially expressed proteins related to thymus ageing,which are mainly involved in energy metabolism processes such as citric acid Salt cycle(TCA cycle),cell growth and death process(cell cycle,apoptosis process),genetic information processing process(splicesome),endocrine system(adipocyte factor signaling pathway),immune system and other pathways.Through protein network interaction analysis,the important differential genes related to thymus ageing such as Fgf2,Flt1,BMP4,v WF,Lamc1,CD19,Cxcr5,and differential protein such as CKS1,SRSF2,CASP-3,FGF2,CPT1 B,PTMA,NASP were screened out.5.Analysis of spleen transcriptome and proteomics results Based on the RNA-Seq method,28 differentially expressed genes related to spleen aging were screened out,which are mainly involved in inflammatory response and immune-related pathways,including chemokine signal transduction pathway,MAPK signal pathway,Rap1 signal pathway and other signal pathways.TMT technology identified 133 tissues with differentially expressed proteins related to spleen aging,which are involved in cell growth and death(cell cycle),genetic information processing(translation,ribosomes),cell motility(actin cytoskeleton regulation),and immunity System and other channels.Through protein network interaction analysis,the important differential genes related to spleen age,such as vsig4,cfh,c7,cx3cl1,penk,ccl8,prkcz,as well as differential protein such as CCNA2,ATOX1,CCR6,CXCL5,EIF3 L,GZMA,RABL3,SLAMF1,RSL24D1 were screened out.Conclusion With the increase of age,the tissue structure and distribution of immune cells in the thymus and spleen of the immune organs of mice have changed,mainly manifested as the destruction of the microenvironment and the decrease of T lymphocytes.The level of inflammatory factors in aging individuals is higher than that in the juvenile group.There are obvious differences in gene and protein expression in thymus and spleen tissues of mice of different ages.In the process of aging,the changes of the thymus at the gene level are manifested as the influence of the thymus microenvironment,the enhancement of adipogenesis,the differentiation and production of thymic epithelial cells,the migration,proliferation or differentiation of thymus cells,and the enhancement of pro-inflammatory effects;At the level,it is manifested as increased energy metabolism,impaired cell growth,genetic material transfer related processes,increased cell apoptosis activity,decreased apoptosis inhibition,enhanced lipid metabolism,affected thymus microenvironment,and increased secretion of pro-inflammatory cytokines.In the process of aging,the changes in the spleen at the gene level are manifested as increased inhibitory activities of T cell proliferation,increased activities that promote cell death,activation of signal transduction pathways for cell apoptosis,and increased pro-inflammatory activities.At the protein level,cell cycle and protein synthesis are impaired,cell proliferation activity is reduced,migration process is affected,antioxidant effect is enhanced,T and B cell activation is enhanced,and pro-inflammatory response is enhanced.The effects of aging on the thymus and spleen mainly include affecting the normal structure of their tissues and their normal growth and development processes.In addition,the enhancement of pro-inflammatory response may be related to the inflammatory state of immunosenescence.The results of this study provide a theoretical basis for elucidating the aging molecular changes of the thymus and spleen and exploring their possible role in immunosenescence. |
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