| Dendritic cells (DCs) are the most potent antigen presenting cells in vivo. In the past decade, most of researchers were interested in how DCs prime the immune response , however, recent studies show that DCs not only can prime the immune response, but also have the ability to down-regulate the immune response or induce immune tolerance. It has been accepted that DCs play a central role in the process of priming naive T cells and tolerance-- central and peripheral tolerance- induction. Especially more and more attention is paid to the function of DCs in the induction of peripheral tolerance. More recently, some kinds of DCs with negative regulatory functions have been reported and the inverstigation of regulatory DCs is one of hot topics in the field of DC immunobiology.The regulatory DCs commonly used in most of the work are prepared in vitro using the immunosuppressive cytokines, such as IL-10 and TGF-β. However, these methods were too simplified to reflect the actual differentiation of regulatory DCs in vivo and the natural counterpart population in vivo.The development of hematopoietic cells in vivo occurs in the context of microenvironment or niche, which consists of many types of stromal cells, such as fibroblasts, macrophages, endothelium cells and adipose cells, etc. The microenvironment gives the exogenous signaling for hematopoietic cells development. Different organs have their special microenvironments, and even in the same organ different compartments have unique sub-microenvironments. Different microenvironments support different type of cell differentiation, for example, the function of the microenvironment in bone marrow. So conclusions of hematopoietic cell development apart from microenvironment cannot reflect the actual situation in vivo.Spleen is the biggest peripheral lymphoid organ, and also it is a hematopoietic organ in mammal. Murine spleen keeps hematopoietic function throughout life. Classical experiment of spleen node conformation proves that microenvironment of spleen supports hematopoiesis, furthermore,microenvironment of different region supports the development of different hemapoietic cells. The hematopoietic stem cells(HSC) are found in spleen of adult mice. So it is conceivable the HSC present in spleen may differentiate into some kinds of cells in the context of splenic microenvironment. Furthermore, the development is a de novo pathway.Splenic stromal cells cultured in vitro could mimic the microenvironment in vivo to some extent, despite their differences in some constituents. There are evidences that long-term cultured splenic stromal cells system can produce dendritic-like cells in the absence of exogenous cytokines. The dendritic-like cells have the phenotype and function of DCs. All of above evidences prove that splenic microenvironment could induce hematopoietic progenitors to differentiate into DCs. However, no one ascertains whether this kind of DCs exists in the spleen in vivo and nobody identifies this DCs' definite function up to now.Generally, stromal cells cultured in vitro consist of multi-components. Purification of these constituents will help to study the role of specific cell type in the induction of DCs. We established the method of preparing endothelial splenic stroma cells (ESSC) from tissue fragment and identified the characters of ESSC. Based on the experimental system of splenic stroma, we found that mature DCs proliferated and differentiated into regulatory DCs in the presence of ESSC. Then we wondered whether ESSC could induce the gerenation of regulatory DCs de novo. The discovery of DCs common progenitors was based on our limited knowledge of DC subsets. We cannot confirm whether there exist other DC subsets and whether the known common progenitors can develop to other DC subsets or not. So the primary aim of this study is to observe whether ESSC can induce differentiation of hematopoietic stem cells towards regulatory DCs.Firstly, we sorted Lin Flt3 CD117~+Sca-1~+ HSCsfrom murine bone marrow by FACSDiva, and cultured them with ESSC for at...
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