Beta-glucan To Promote Bone Marrow Mononuclear Cells To Secrete Angiogenic Growth Factor

: To investigate the enhanced production of angiogenic growth factors by bone marrow mononuclear cells activated withβ-glucan and its possible mechanism. Bone marrow mononuclear cells were harvested from tibia and femur of six male Lewis rats about 280g-320g using density gradient centrifugation and white blood cells lysate. These cells were incubated in RPMI-1640 containing 1% fetal bovine serum (FBS) medium with or withoutβ-glucan (10ug/ml and 100ug/ml). Cells were seeded into 24-well tissue culture plate (1x107cells/well) and incubated at 37℃in a humidified incubator in normoxic (95%O₂/5%CO₂) and hypoxic (1%O₂/5%CO₂) condition to allow the cells activated byβ-glucan. After 22 hours incubation, the medium containing non-adherent cells were removed by washing two times with serum-free RPMI-1640, and the cells were refed with RPMI containing 1%FBS and incubated additional 48 hours in normoxic condition. VEGF protein in conditioned media was assayed using a sandwich ELISA kit, following the manufacturer's protocol. Conditioned media was added to cultured human umbilicus vein endothelial cells (HUVEC) to assess the effect of BM-MNCs on HUVEC. At the same time, we got RNA from these cultured cells to analysis RT-PCR. In ELISA, the level of VEGF was significantly enhanced with 10ug/mlβ-glucan in normoxic and hypoxic condition; In control group, the level of VEGF in hypoxic condition was obviously increased than that in normoxic condition; In some extent, the level of VEGF was not enhanced with the increased concentration ofβ-glucan. The mRNA expression of VEGF with 10ug/mlβ-glucan was significantly enhanced under hypoxia by RT-PCR analysis. In MTT assay, the effect of conditioned media (10ug/ml and 100ug/mlβ-glucan) on the viability of HUVEC cells was increased in hypoxia. The effect ofβ-glucan on the production of angiogenic growth factors secreted by bone marrow mononuclear cells was first studied. VEGF mRNA expression and protein synthesis were upregulated byβ-glucan stimulation especially under hypoxic condition. The culture supernatant ofβ-glucan-stimulated BM-MNCs clearly enhance the proliferation of HUVEC cells. BM-MNCs activated withβ-glucan enhanced the production of angiogenic growth factors, which helps to angiogenesis, and affords a new method to ischemic diseases treatment.