Study On The Gene Expression Patterns In Chick Thymus During Postnatal Development

The thymus is the site for T cell development,differentiation and maturation.The T-progenitor cells are generated in the bone marrow.These progenitor T-cells enter the thymus microenvironment under the influence of various chemokines,where they develop and differentiate into mature T cells and then migrate to the peripheral blood and lymphoid organs and maintain the body health.However,the animal's thymus experiences age-related atrophy,which in turn decreases its function,and consequently the body becomes senescent and diseased.Therefore,understanding the basic principles of animal thymus development,exploring the mechanism of its atrophy and knowing the effective methods to enhance its function and inhibit its degradation has very important practical significance for the prevention and diagnosis of diseases and resistance to immune related aging.At present,there are a few reports on gene expression changes and molecular mechanisms in the growth and development of chicken thymus.Therefore,in this study,broiler chickens?Cobb strain?were used as research objects.Several diagnostic research techniques such as HE,toluidine blue and Immunohistochemical staining,RNA-Seq,as well as real-time fluorescence quantitative PCR were used to study the alterations in the tissue micro-structure and the gene expression changes during chicken thymus growth and development.The main research contents and results are as follows:1.Changes of weight and index during chicken thymus developmentThe body weights and thymus weights of 0,1,5,9,18 and 27 weeks old chickens?n=6?were determined and the thymus index?thymus weight?g?/body weight?kg??was calculated.With the increase in age,the weight of thymus increased first and then decreased,however,it was maximum at 18 ^th week while atrophy seriouly at 27 ^th week.From 0w to 1w,the thymus index showed an upward trend,and from 1w to 27w,the thymus index showed a downward trend.2.Changes in tissue micro-structure during chicken thymus developmentParaffin sections of chicken thymus tissues at different age-stages?in weeks?were stained with HE.The results showed that the thymus was divided into the outer cortical and the inner medullary areas.The cortex was mainly composed of lymphocytes and a small amount of epithelial cells with deeper coloration,the medulla was mainly composed of epithelial cells and a small amount of lymphocytes with lighter coloration,which has a typical thymus body structure.With the increase in age,the cortex became thinner,the medulla increased in area,and the cortic-medullary area ratio was gradually decreased,the blood vessels and Hassall's body in the thymus tended to increase.3.Changes in cell proliferation during chicken thymus developmentThe thymus tissue sections of chickens at different age-stages?in weeks?were stained by immunohistochemistry to detect the distribution and developmental changes of PCNA positive cells.The results showed that PCNA-positive cells?proliferating thymic cells?were distributed throughout the thymus and gradually decreased with the advancement of chicken age.The results showed that cell proliferation in chicken thymus gradually decreased with the increase in age.4.Changes in mast cells during chicken thymus developmentToluidine blue staining was performed on the chicken thymic tissue sections of different age stages in weeks to examine the distribution and developmental changes of mast cells.The results showed that mast cells were mainly located in the medulla of the thymus and around the blood vessels.The number of mast cells increased at 1 ^st week and gradually decreased after 9 ^th week.5.Changes in gene expression during chicken thymus developmentRNA-Seq and data analysis were performed to study gene expression patterns during chicken thymus development.After sequencing,the original data was21723697300bp.After filtering the low-quality reads,the clean data was 21708736650bp,the clean reads were 434174733,the average of clean reads were 24120819,and the overall clean reads rate was all greater than 99.9%.The comparison rate with the reference genome reached 90%.Quantitative analysis of gene expression was performed.Expression trends were analyzed on the basis of gene expression levels,a total of 10trends were significantly enriched.The trend of continuous up-regulation and down-regulation was used for GO and KEGG pathway enrichment analysis.Profile 39?i.e.,up-regulation trend?GO enrichment analysis results showed that it was mainly related to inflammation,immunity,signal transduction and apoptosis.The KEGG enrichment results were mainly associated with the immune system,secretory system,infectious diseases,signaling,cellular activity and lipid metabolism.Profile 8?i.e.,down-regulation trend?GO enrichment analysis results showed that it was mainly related to the composition,transmission,repair and energy metabolism of genetic material.The KEGG enrichment analysis results were mainly associated with cell growth and death,genetic information transmission,energy and carbohydrate metabolism and amino acid and nucleotide metabolism.Several key genes such as PCNA,CDK1,CCNA2 and CCNB2,which are closely related to cell proliferation and genetic information transmission in down-regulated cell cycle pathways,were tested by fluorescence quantitative PCR.The results showed that their changes were consistent with the sequencing results,the amount of expression continues to decrease over time.The above results indicate that with the development or ageing of the thymus,cell proliferation is reduced,the thymus gradually destroyed due to severe changes in the thymic microenvironment,leading to reduced thymus weight.The transmission of genetic material in the thymus is disrupted,the level of metabolism in the thymus is reduced,fat metabolism is increased,inflammation and immune response are enhanced.