The Effects Of CCL3on Osteoblast Inhibition In Myeloma-induced Bone Disease

ObjectiveTo culture osteoblast in vitro from bone marrow of mutiple myeloma patients and explore their biological characteristics and osteogenic potential.To evaluate CCL3receptor CCR1expression levels in MM patient-derived OBs and address the effects of CCL3on osteoblastogenesis.MethodsFirst section:Ten MBD patients and8healthy donors as controls were enrolled in this section. Osteoblasts from BM of MBD patients were cultured in vitro with dexamethasone,β-sodium glycerophosphate and vitamin C,and identified by ALP staining,Von Kossa’s staining. The antigens(CD34-CD138-CD45-)on osteoblasts were examined with flow cytometric assay. The generation and osteogeniec potential of osteoblasts from MBD patients and normal subjects were compared.The CCL3receptor expression were evaluated by Flow cytometry too.Second section:Twenty-one MBD patients were enrolled in this section. Osteoblasts from BM were cultured in vitro and identified by ALP staining,Von Kossa’s staining. At the passage3, OBs were divided into three groups:blank group(group Ⅰ),CCL3group which OBs were cultured with CCL3(group Ⅱ).The group Ⅲ were treated with CCL3and a neutratizing antibody against CCL3. The changes of their osteogenic potential and biological characteristics were observed after intervene.The levels of osteocalcin(OCN) in supernatants of OBs were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA expression of RUNX2、Wnt and Osterix were analyzed by quantitative-PCR.ResultsFirst section:Osteoblasts from BM of MBD patients could be cultured in vitro with dexamethasone,β-sodium glycerophosphate and vitaminC.The shape of the cells was almost spindle and polygon.The double time was about22hours in MBD patients and20hours in normal control.ALP staining,Von Kossa staining were positive to verify the osteoblasts. The number of the positive cells and depositions of MBD patients was less than that of normal controls. MM-derived OBs express higher levels of CCR1(74.48±7.31)%compared with normal controls’OBs (48.35±8.81)%.Second section The CCL3did not affect morphology and quantity of OBs,but matrix mineralization was significantly impaired in OB cultures exposed to CCL3.The average levels of OCN detected in72-h culture supernatant of OBs exposed to CCL3was (750.643±41.116)μg/L,which is lower than that in blank group[(803.375±40.654)μg/L] and increased by CCL3antibody in the presence of CCL3[(787.358±32.063)μg/L)].CCL3treatment downregulated both Runx2and Osterix mRNA expression(0.4002±0.3734±0.5242±0.2809,respectively).And the inhibition was partly reversed by the trentment with anti-CCL3.The expession level of Wnt mRNA after exposure to CCL3showed a downward trend(0.7341±0.4432),but showed no statistical significance.Conclusion:Osteoblasts could be cultured in vitro from BM of MBD patients.The proliferation and osteogenic potential of osteoblasts from MBD patients were decreased.CCL3play a important role in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function.