| Background and objective: All-trans retinoic acid(ATRA), a main active metabolite of retinol (Vitamin A),participate in the regulation of proliferation differentiation and apoptosis of a variety of cell types.Its influence on immunological system , especially the inhibitory effect on negative selection ,may become a new focus to be investigated. Negtive selection plays a very important role in maturate of T cell. And the mechanism is the induction of apoptosis of the auto-reactive immature T cells by autoantigen, which is presented by APCs(antigen-presenting cells) in thymus.This process makes I cells obtain immunological tolerance to autologous tissues. It has been proved that the abnormality of negative selection will result in autoimmune diseases. IDDM is an autoimmune disease in which P cells are destroyed. There are infiltrations of lymphocytes and monocytes in islets, and the infiltrating lymphocytes are mainly T cells. This suggests that autoimmun mediated by T cell may play an important role in the occurance Qf IDDM.On the other hand,it has been reported that there is abnormality in metabolism of retinol in IDDM,which may result in the increase of the concentration of ATRA.So it is of significance to study how negative selection is regulated and to investigate itsi possible role in IDDM.Now, in the research about the relationship between ATRA and negative selection ,anti-CD3 monoclonal antibody-induced apoptosis of thymocyte is used as a model for elucidating the later in vitro. The model is simple, but it may be quite different from the real mecbanism.Thus, in present study, we established a new model in which superantigen(SEB),with the assistance of APCs cultured in vitro,was used to stimulate thymocyte ,and hoped it could elucidate the effect of autoantigen in negative selection.To determine whether the model was success,we carried out the qualitative and quantitative analysis of apoptosis of the thymocyte. Then we made a further investigation into the influence of ATRA on negative selection. Finally, we made a measurement of the concentration of ATRA in IDDM patients. The objective is to study the relationship between ATRA and the occurence of IDDM. Methods and results: 1 Establishing the model of negtive selection. (l)Thymic stromal cell and thymocyte were isolated and mix-cultured routinely and then divided into two groups: group SEB (receiving SEB 1 ~.i g/ml) and control.(2)Apoptotic analysis of thymocyte: (a) Agarose gel electrophores of DNA: there was characteristic DNA ladder in group SEB,but not in control.(b)Hoechst 33258 fluorescent staining: there were much more cells which were stained densely and unevenly in group SEB than in control,and the rate of apoptotic cells in group SEB(56.0 6.9%) was higer than that in control(8.7 2.8%),P < 0.0 l.(c)Assay of DNA fragmentation with DPA: DNA fragmentation was higher in group SEB(61.4 0.0%) than in control(8.2 .3%),P K 0.0l.(3)Expression of Fas and FasL was detected by flow cytometry: Fas expression in group SEB(58.4 .6%) was higher than in control (11.1 .7% );FasL expression in group SEB (47.7 .8%) was also higher than in control(9.5 .7%).P |
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