The Effect And Mechanism Of SPARC On The Development Of Hematopoietic Cells

The study of SPARC function was mainly focused on the growth, metastasis and prognosis of some cancers, whereas it’s role in hematopoiesis was rarely reported. The expression of SPARC was decreased in bone marrow (BM) of patients with5q deletion myelodyspastic syndrome (5q-MDS). Immuunomodulator lenalidomide was used to treat the patient with5q-MDS, which could inhibit the proliferation of malignant erythroblasts, meanwhile the expression of SPARC increased. In addition, SPARC-null mice showed a lack of immune response for footpad lipopolysaccharide challenge, meanwhile the percentage of CD19+B lymphocytes was decreased in BM. These data implied that SPARC was involved in erythropoiesis and B lymphopoiesis. In this study, SPARC-null mice were used to study the role of SPARC in erythropoiesis and B lymphopoiesis. This study includes three parts as follows:Chapter one The effect and mechanism of SPARC on the development of erythrocytesObjective:Analyze the development of erythrocytes in SPARC-null mice, and explore the mechanism of SPARC affected erythropoiesis. Investigate the effect of SPARC on the development of human erythrocytes.Methods:Blood analyzer, flow cytometry, colony-forming assays and BM transplantation were used to analyze the development of erythrocytes in SPARC-null mice. Magnetic beads sorted cord blood CD34+cells, colony-forming assays and erythroid differentiation were used to investigate the effect of SPARC on the development of human erythroid.Results:1. RBC count in peripheral blood and the percentage of Ter119+cells in BM of SPARC-null mice were unchanged compared with wild-types. However, SPARC-null bone marrow cells (BMCs) gave rise to less number of erythroid brust colony-forming units (BFU-E) and spleen colony-forming units (CFU-S8) than wild-types, suggesting that SPARC deficiency inhibited the development of erythroid progenitors.2. Long-term cultured SPARC-null BMCs, gave rise to less number of CFU-GM and BFU-E than wild-types. However, BM transplantation showed that SPARC deficiency did not affect the potential of hematopoietic cells differentiated into erythroid, but impaired the ability of hematopoietic microenvironment supported the development of erythroid progenitors.3. SPARC promoted cord blood CD34+cells formed CFU-E and BFU-E. During erythroid differentiation, Hb content was increased by the addition of SPARC, but the percentage of CD71+GPA-and CD71+GPA+cells was unchanged.Conclusion:SPARC promotes the development of erythroid progenitors, but not affect terminal erythroid differentiation.Chapter two The effect and mechanism of SPARC on the development of B lymphocytesObjective:Analyze the development of B lymphopocytes in SPARC-null mice, and explore the mechanism of SPARC affected B lymphopoiesis.Methods:Flow cytometry, BM transplantation and in vitro B-cell differentiation, cell proliferation and apoptosis analysis were used to study the effect of SPARC on B lymphopoiesis. Real-time RT-PCR, Western blotting and Gene chip were used to explore the mechanism of SPARC on B lymphopoiesis.Results:1. The percentage of B-cell was significantly increased in PB of SPARC-null mice, but pro-B, pre-B and immature B cells decreased in the BM.2. The percentage of Lineage-Sca-1+c-kit+(LSK) cells was significantly decreased in BM of SPARC-null mice. However, BM transplantation showed that SPARC deficiency did not affect the potential of hematopoietic cells differentiated into B lymphocytes, but impaired the ability of hematopoietic microenvironment supported the development of B lymphocytes.3. SPARC-null BMSCs inhibited B-cell differentiation. BMSCs derived from wild type mice expressed SPARC, but the addition of recombinant SPARC did not affect B lymphocyte differentiation on SPARC-null BMSCs.4. SPARC deficiency did not affect the expression of IL-7and SDF-1in BMSCs, but conditioned medium of SPARC-null BMSCs inhibited B-cell differentiation, suggesting that this conditional medium contains soluble inhibitors. Gene chip analysis showed that serine proteinase inhibitor serpina3n and MMP9were highly upregulated in SPARC-null BMSCs compared with wild-types, but both of them did not contribute to the reduction of B lymphopoiesis in SPARC-null mice.Conclusion:SPARC deficiency in mice impaired B lymphopoiesis, but SPARC had no direct effect on B lymphopoiesis. SPARC deficiency altered BMSCs released soluble factors, indirectly regulated the development of B lymphocytes.Chapter three The effect of adipocytes on the development of B lymphocytesObjective:The number of adipocytes was increased in BM of SPARC-null mice, and adipocytes negatively regulated the development of hematopoietic cells. We presume that the increased adipocytes may partly account for the impaired B lymphopoiesis in SPARC-null mice. In this chapter, we studied the effect of adipocytes on the development of B lymphocytes.Methods:To study the effect of adipocytes on development of B lymphocytes. OP9cells were differentiated into adipocytes, and then BMCs from C57/BL mice was cocultured with OP9-dericed adipocytes for B-cell differentiation.Results:1. OP9cells could rapidly and efficiently differentiate into adipocytes, and OP9-derived adipocytes expressed white adipocyte tissue (WAT) specific genes adiponectin and leptin, brown adipocyte tissue (BAT) specific genes Dio2and PRDM16.2. OP9-derived adipocytes and their conditioned medium inhibited B-cell differentiation.3. OP9-derived adipocytes not only inhibited the formation of B lymphocyte progenitors, but also inhibited the transition of pro-B to pre-B cells.Conclusion:Adiopocytes inhibited B-cell differentiation, which may partly account for the decline of B lymphopoiesis in SPARC-null mice. The paper includes26figures,19tables, and71references.