Study On The Effect Of Abnormal Hedgehog Signal Of Osteoblast Lineage On The Development And Biological Characteristics Of Dendritic Cells

Objective:More and more studies have found that osteoblasts play an important role in the development and regulation of hematopoietic stem cells.Once osteoblasts are damaged,the hematopoietic phenotype that leads to skeletal abnormalities and myeloproliferative in mice,it mainly includes the clonal proliferation of HSCs,decreased blood cell counts,abnormal B lymphocyte production and megakaryocyte development,which will eventually cause some diseases,such as acute myeloid leukemia(AML).Dendritic cells are currently the cells with the strongest antigen-presenting ability,and DC play an important role in both adaptive immunity and innate immunity.But so far,the differentiation and development of dendritic cells by the osteoblast lineage has not yet been known.This topic aims to explore the influence of osteoblast lineage on the development and biological characteristics of dendritic cells and its molecular mechanism.Aim:(1)To study the role of the negative regulator sufu in the Hedgehog pathway in mice in regulating bone metabolism through conditional gene knockout technology;(2)To explore the proportion and biological characteristics of dendritic cells(DC)in the bone marrow,spleen and lymph nodes of mice conditional knocked out sufu(Osx-Cre;sufufl/fl),and to explore its mechanism preliminarily.(3)To Analyze the molecular mechanism of the abnormal Hedgehog signal in the osteogenic lineage affecting the differentiation and development of DC.Methods:(1)Construct conditional knock-out sufu in osterix mice(Osx-Cre;sufufl/fl),we compare the body size of the wide type(control group)mice(Osx-Cre)and conditional knock-out mice(Osx-Cre;sufufl/fl),analyze the bone quality by Micro-CT scan,and detect whether the mice bone quality is abnormal by H&E staining;qRT-PCR and WB were used to detect the expression of genes or proteins related to osteogenic differentiation of bone tissues,and to analyze the proportion of osteoblasts in the bone marrow by flow cytometry.(2)Construct R26-mTmG and Osx-Cre;R26-mTmG mice and observe the distribution of osterix+ cells in bone tissue,spleen and lymph nodes;Flow cytometric analysis of the proportion of dendritic cell(DC),conventional DC(cDC)and plasmacytoid DC(pDC)in the bone marrow,mouse spleen and lymph nodes of the control group(Osx-Cre)and conditional knock-out mice(Osx-Cre;sufufl/fl),and count it;cDC and pDC in the bone marrow are sorted and activated,co-cultured with CD4+T cells,and qRT-PCR is used to detect the difference in biological characteristics of cDC and pDC;Flow cytometry and in vitro induced dendritic cells were used to detect differences in the proportion and function of hematopoietic stem cells in bone marrow.(3)Gene sequencing to detect the difference gene expression between the control group and conditional knock-out mice of BMSCs;qRTPCR and WB detect the expression of WIF1 in the bone tissue of the control group and knock-out mice.Flow cytometry and qRT-PCR to detect the expression of β-catenin in c-kit cells in the bone marrow,and to detect the inhibitor of Wnt/β-catenin signal(ICG001)influence on the proportion of induced dendritic cells in vitro and to explore the relationship between Hh signaling and Wnt/β-catenin signaling pathway.Results:The size and weight of Osx-Cre;sufufl/fl mice were significantly lower than those of control group mice(Osx-Cre),the results of Micro-CT scan and H&E staining analysis showed that compared with the control group mice,Osx-Cre;sufufl/fl mice had severe osteoporosis;The bone tissue of qRT-PCR and WB results showed that the expression of osteogenic differentiation genes and proteins in the bone tissue of Osx-Cre;sufufl/fl mice were significantly lower than those in the control group mice.Flow analyzing found that the proportion and the number of osteoblasts in the bone marrow of Osx-Cre;sufufl/fl mice is significantly lower than that of control mice.Flow cytometry analysis showed that the proportion and number of osteoblasts in the bone marrow of Osx-Cre;sufufl/fl mice were significantly lower than those of control group mice.(2)A large number of osterix cells were observed in the bone marrow through slices,and certain osterix+cells were also present in the medulla of the spleen,while a small amount of osterix+cells were only present in the cortex of the lymph nodes;Flow cytometry results showed that compared with control group mice,the proportion and number of pDC in the bone marrow and spleen of Osx-Cre;sufufl/fl mice decreased,and the proportion of cDC in the bone marrow also decreased significantly;Activated cDC and pDC in the bone marrow of the control group and the Osx-Cre;sufufl/fl mice were co-cultured with CD4+T cells.The results showed that the function of cDC in the bone marrow of the control group mice was no different from that of Osx-Cre;sufufl/fl mice,but the ability of pDC in the bone marrow of Osx-Cre;sufufl/fl mice to induce CD4+T cells was significantly weaker than that of the control group mice;The qRT-PCR results of cDC and pDC in the bone marrow showed that the genes related to the DC differentiation of cDC and pDC in Osx-Cre;sufufl/fl mice were less than those in the control group mice,and the expression of function-related genes was also lower than that in the control group mice.The proportion of hematopoietic stem cells in the bone marrow of Osx-Cre;sufufl/fl mice was significantly lower than that in the control group mice.Control group mice can get more DC cells induced than Osx-Cre;sufufl/fl mice in vitro,but there is no difference in the ratio between its subgroups(CD11b+DC and B220+DC),and there is no difference in function of DC induced from control group and Osx-Cre;sufufl/fl mice.(3)Gene sequencing results show compared with the control group,the bone tissue of Osx-Cre;sufufl/fl mice highly expresses WIF1 gene and protein.The expression of β-catenin in c-kit+cells and the expression of Wnt/β-catenin-related genes in the bone marrow of Osx-Cre;sufufl/fl mice were significantly lower than those of the control group mice.Wnt/β-catenin(ICG001)does not inhibit the generation of DC,but it obviously inhibits the generation of pDC.qRT-PCR and WB detection showed that the inhibition of Hh signal promoted the activation of Wnt/βcatenin signaling pathway.Conclusion:(1)Conditional knocking out the sufu gene in osterix+cells resulted in abnormal osteogenesis in mice,which leads to severe osteoporosis in mice.(2)The proportion and function of pDC in the bone marrow of Osx-Cre;sufufl/fl mice,and the proportion of pDC in the bone marrow of Osx-Cre;sufufl/fl mice are abnormal,and the proportion of HSCs in the bone marrow is abnormal.(3)Bone marrow mesenchymal stem cells of Osx-Cre;sufufl/fl mice highly express WIF1,leading to an increase in WIF1 in the bone marrow microenvironment,inhibiting the Wnt/β-catenin signaling pathway of hematopoietic stem cells,and reducing the production of pDC.