| Background and Objective: As we all know, hematopoietic stem cells are a rare population of primitive cells able to give rise to all mature blood and immune cells, and have the capacity of self-renewal and multi-differentiation. HSCs transplantation is an effective treatment for varieties of malignant diseases, such as leukemia, autoimmune disorders and so on. However, the high cost and insufficient HSCs supply limited the clinical application of this treatment. How to efficiently amplify a large number of HSCs in order to meet the clinical need and lower the coat has become an urgent issue for HSCs transplantation.It has been proved that FGF signaling pathway plays a major role in stem cell self-renewal, maintenance, and development, as well as in bone marrow recovery. In vivo, FGF2 administration successfully expanded both MSPCs and HSPCs. FGF1 signaling was shown to be important for enhancing 5-FU-mediated bone marrow post-injury repair and AMD3100-induced HSPCs mobilization. FGFR3 is an important receptor of FGF. It has been proved that the expression level of FGFR3 is high in bone marrow. However, the effect of FGFR3 on the HSCs proliferation is not yet clear. The aim of this article is to explore the regulatory role of FGFR3 in HSCs proliferation in vivo.Method: 1. Firstly we established Fgfr3 knockout mice. 2. We mainly used trypan blue staining assay for the total cells counting. Depending on the cell surface markers, we analyzed the proportion of LSK cells in bone marrow and spleen of WT and KO mice by flow cytometry. Then we further analyzed the total number of LSK cells. 3. Brd U labeling was used to detect the proliferation of HSCs. 4. We detected the expression of FGFR1 by flow cytometry. 5. By intracellular staining we detected phosphorylation levels of NF-κB and STAT1. 6. The expression level of p15 m RNA was detected by RT-PCR. 7. Through bone marrow transplantation, we explored the impact of FGFR3 on hematopoietic reconstitution and the ability of HSCs differentiation.Results: 1. At steady state, there was no difference in the total cells and LSK cells in bone marrow between WT and KO mice. 2. Differentiation level of HSCs into neutrophils(Gr1 + /CD11 b +), B cells(B220 +) and T cells(CD3e +) in bone marrow also showed no difference between WT and KO mice. 3. On the 14 th day post treatment with the chemotherapeutic agent 5-FU, the total HSCs in KO mice bone marrow and spleen were 2.1- and 1.8-folds of that in WT mice, respectively. 4. Brd U positive rate of HSCs in KO mice was 1.5-folds of that in WT mice. 5. Flow cytometry results showed that, after the administration of 5-FU, the expression level of FGFR1, p-NF-κB of HSCs in KO mice was significantly increased. 6. We observed down-regulation of p-STAT1 in KO mice after the administration of 5-FU. 7. The expression of cell cycle suppressor gene p15 decreased at m RNA level. 8. The hematopoietic reconstitution ability and differentiation of HSCs in vivo presented no differences after bone marrow transplantation between WT and KO mice.Conclusion: We concluded that there are two FGF-FGFR signaling pathways affecting HSCs proliferation. One is FGF1-FGFR1 signaling pathway which has already known to be the promoter of HSCs proliferation; the other is FGF1-FGFR3 signaling pathway, which is proved to induce inhibition of HSCs proliferation by this study. In this article, we presented the first in vivo evidence showing that FGFR3-mediated signaling pathway negatively regulates HSCs proliferation in response to severe BM damage. |
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