| Objectives It is a serious problem yet impairing the health of children in impoverished area that Vitamin A deficiency leads to the depression of immunity. Retinoic acid (RA) is the active metabolite of vitamin A, which can increase the production of antibody from B cells, but the mechanism of its effect remains un-completely clear. Lymph node, as the most important peripheral immune organ, where B cells respond to immune antigens and produce antibody, is the important site for B cells to further differentiate and maturate. In order to further clarify the mechanism of retinoic acid promoting the production of antibody, we investigated the effect of retinoic acid on the differentiation and maturation of B cells from lymph node of children, and relational changes of the expression levels of retinoic acid receptor genes in B cells of lymph node. The study will provide theoretical base for the reasonable supply of vitamin A to reinforce the antiinfective immunity in children.Methods From 32 patients with digestive tract malformation underwent intra-peritoneal operation in the surgical ward of our hospital, the lymph nodes in the margin of excised tissues were obtained. 6 Digoxin labeled antisense RNA probes for retinoic acid receptors mRNA(RARα,β,γ and RXR α.β.γ)were used, and in situ hybridization was performed on the frozen successive sections of lymph nodes, to observe the expression and distribution of retinoic acid receptor genes in lymph nodes. The expression of retinoic acid receptors in mRNA level was quantitatively analyzed by reversed transcription and fluorescent quantitative PCR. Cells separated from lymph nodes were divided into 5 groups: Retinoic acid (RA), Retinoic acid plus Ro 41-5253, retinoic acid receptor antagonist (RA+Ro), lipopolysaccharide (LPS), lipopolysaccha-ride plus Retinoic acid (LPS+RA) and control cultured in vitro. After 24h and 48h of cell culture, the surface markers on the cells were detected by flow cytometer to observe the maturation and activation of B cells. At the end of cell culture, the cells were harvested and total RNA was isolated, the expression levels of retinoic acid receptor genes were quantitatively analyzed by RT - fluorescent quantitative PCR.Results (1)in situ hybridization showed that all the 6 retinoic acid receptor genes were expressed in lymphocytes and reticular cells of lymph node, the distribu-tion was widespread. (2)RT and fluorescent quantitative PCR showed a varying expression of 6 retinoic acid receptor genes in lymph node among different age children, the mRNA levels of all receptor genes was lower in children younger than 1 year, and then increased gradually with the development of the immune system. ?During the culture, the percentages of relatively mature CD19+IgM+ B cells were increased and relatively immature CD19+IgM" B cells were decreased gradually, the changes were especially obvious at 48 hours. The administration of retinoic acid further increase the percentages of CD19+IgM+ B cells, compared with the control (without retinoic acid), the enhancement was marked at 24 and 48 hours (P<0.05). However, when the retinoic acid receptor antagonist was added to the culture, the promotion effect of retinoic acid was completely counteracted. ?In vitro culture, after administration of atRA, the mature IgM+IgD+CD25" B cell was obviously increased compared with the control (P<0.05), whereas, the immature IgM+IgD" CD25"B cell was obviously lower than the control (P<0.05), and the difference was more obvious at 48 hour (P <0.05). ?When RA was added into the cell culture together with LPS, the activated B cells (IgM+ CD25+) was markedly increased compared with the LPS stimulation alone (P<0.05), but the rest B cells (IgM+ CD25') was obviously decreased (PO.05). All these influences were more obvious in children younger than 3 years. Moreover, RA also markedly increased the expression of RARa gene in B cells during their maturation and activation, whereas RARa antagonist can effectively counteracted the promotion of RA on B cells and the up-regulation of RARa mRNA.Conclusions: The expression of retinoic acid receptor genes in lymph nodes of children was abundant, widespread, and varied with age. RA can promote the development and activation of B cell from lymph node in vitro cultured. The retinoic acid receptor was the main pathway to mediate the actions of RA. The effect on B cell of lymph nodes may be the important mechanism of vitamin A reinforcing the anti-infective immunity in young children. |
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