| [Background] Many tissues and organs contain mesenchymal stem cells (MSCs), and the field of regenerative medicine have a great amount of interest in the MSCs due to their unique biological properties:(1) they have the ability to differentiate into different cell types;(2) they are lack of immunogenicity and they show the ability to perform immunomodulatory functions;(3) the little teratoma formation. Previously, we isolated the Sca-1+MSCs from mouse bone marrow by the stem cell marker, stem cell antigen-1(Sca-1)----they have osteogenic differentiation and adipogenesis capacity, and have immunomodulatory abilities. Additionally, Sca-1+MSCs could make hematopoietic progenitor cells (HPCs) to develop into regulatory dendritic cells (DCs), and we found MSCs can influence the fate of DCs by studying the biological characteristics such as cell morphology, phenotype and the functions of the new DCs. However, we do not know much about its mechanism of the induction. In this thesis, we focus on the immunomodulatory ability of Sca-1+MSCs and its mechanisms.[Purpose]1. Sca-1+MSCs’immune regulatory functions are tested in vivo, and we focus on its effects on the differentiation of DCs;2. Compare the different DCs, and searching for the most important transcription factor;3. The function of Notch inhibitor (DAPT) is tested in the generation of the new DCs.[Methods]1. Sca-1+MSC is separated and purified using a magnetic method, and its biological characteristics are tested, including the growth curve, cell cycle, cell phenotype identification, adipogenic and osteogenic differentiations;2imDCs and maDCs are cultured in vitro, and imDCs are co-cultured with Sca-1+MSC to observe the generation of regulatory DCs by flow cytometry, find out the key cytokines in coculture medium using enzyme linked immunosorbent assay (ELISA) methods, and the key transcriptional factor in DC differentiation is tested by Western blotting.[Results]1.The bone marrow of mice has Sca-1+MSCs;2Sca-1+MSC has a very typical feature of stem cells:spindle or spindle-shaped morphology, log phase growth curve, high proportion of G0/G1phase cells, stem cell surface molecules are expressed highly, and they show the ability of adipogenic and osteogenic differentiation.3. Sca-1+MSCs can induce imDC differentiate to CD11bhighlalow regDCs, which are different with imDCs and maDCs in cell phenotype, secretion of cytokine and the expression of cellular transcription factor.4. Sca-1+MSCs suppressed the maturation of imDCs, even LPS cannot change it.5. The secretion of inflammatory cytokines (IL-12, TNF-a) is related to IRF8, and regDCs shows a low expression of IRF8, ELISA results showed reduced secretion of IL-12and TNF-a, and IL-10secretion is high, these results suggesting that Sca-1+MSCs have immunomodulatory effects on DCs.6. When add notch inhibitor (DAPT) into the culture system, the expression of IRF8is up-regulated, and the secretion of IL-12and TNF-a is also increased, these results show that Notch inhibitors (DAPT) reduced the induction of Sca-1+MSCs.[Conclusion] Sca-1+MSC can induce imDCs differentiate to CD11bhighIalowCD11clow DCs, and notch inhibitor (DAPT) can influence this induction. |
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