| We removed spleen of 5 chickens each on the 13th, 15th, 18th, 20th embryonic day, and the 1st, 4th, 7th,14th, 35th, 56th post-hatching day. We made cryosections for the H.E.stain and immunohistochemistry,meanwhile, we took another 5 chickens each time after 20th embryonic day to separate lymphocytes for thelymphocyte transformation test.This study investigated the tissue structure of spleen at different stages of growth and development byHE staining, we found that the white pulp and red pulp began to recognize from embryonic day 18; In thespleen of 4 day-old chicken, periarterial lymphoid sheath(PALS) could be observed obviously; Spleen nodeappeared at 14th post-hatching day; With the increase of age, these characteristic structure were maturegradually, by day 21 after hatching, the boundary and ratio of white pulp to red pulp have been stable. Afterthis time, spleen nodes were even more and bigger, specially at 35 and 56 day-old.In the present study, we have combined immunohistology and computer graphic analytical techniqueto investigate the development processes of T lymphocytes and subpopulation at different age-old. Theresult revealed that the CD3~+ T lymphocyte individually appeared in the spleen from embryonic day 18,these positive cells were main distributed in the PALS and red pulp; With the increase of day-old, thenumber of CD3~+ cells showed the tendency to rise, and was stable at 21 day-old. The CD3 molecules on theCD3-positive ceils were less to express and were still not mature. The appearance time of CD4~+and CD8~+cells were different, CD8~+ cells appeared earlier than CD4~+, with more cells in quantity than the latter oneall the time. With the increase of age, both of them showed the tendency of rising in overall number, theyreached peak value at 21 day-old, and the differences were significant when compared to other age-old (P＜0.05).The CD4 and CD8 molecular on the positive cells expressed stable at 7 day-old, the resultindicated that CD4~+ and CD8~+ cells reached mature level at this time. CD4~+ cells mainly distributed in thePALS, the positive cells were clearly observed in the red pulp until 21 day-old, besides, there were also afew of CD4~+ cells in the spleen node; From the age of 4 day-old, there were many CD8~+ cells in both PALSand red pulp, but no CD8~+ cells distributed in the spleen node all the time. The ratio of CD4~+ cells to CD8~+cells basically keeps from 0.3 to 0.5.We carried out lymphocyte transformation test on lymphocytes of chicken spleen. We investigatedthat T cells from chicken spleen have exhibited the ability to respond to immune stimulation (ConA) from4 day-old, we also found that the lymphocyte from spleen had high spontaneous proliferation activity at 4-7day-old. The stimulation indexes (SI) showed the tendency of rise at different ages, this indicated that theability of T cells from chickens to respond to immune stimulation has been enhanced with the increase of Our research demonstrates that there are close relationships among histological development of spleen,development of T lymphocytes and the development of immune function: the immigration of Tlymphocytes can enrich tissue structure of spleen as PALS; The special structure of spleen can also providefunctionating place for lymphocytes; Likewise, the cytokine are discharged by T lymphocytes, when Tlymphocytes perform immune function, it can stimulate the development of T,B-lymphocytes and theircorrelated structure, for example, PALS, ellipsoid and spleen node. Therefore, they are supplements to eachother and neither can be omitted.To sum up, we study the development of T lymphocytes and their immune function in the chickenspleen, following three levels: tissue level, cellular level and molecular level. Thereby we can understandthe immune state at chicken embryo and immediate post-hatch. This study expands the contents of avianmolecular immunology and pathophysiology, moreover, it can provide rationale for immunity at the rightmoment, pathopoiesis mechanism and disease prevention.
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