多发性骨髓瘤骨病患者成骨细胞培养及CCL3对成骨细胞作用的研究

Effect of CCL3 on osteoblast in myeloma bone disease

目的 建立多发性骨髓瘤（MM）患者成骨细胞体外培养体系,检测成骨细胞膜CCL3受体（CCR1）表达,研究CCL3对MM患者成骨细胞（OB）的作用.方法 采用密度梯度离心法分离骨髓单个核细胞（BMMNC）,应用地塞米松、β-甘油磷酸钠及维生素C诱导并纯化获得MM患者成骨细胞,以碱性磷酸酶染色及Von Kossa＇s钙染色鉴定成骨细胞,用流式细胞术（FCM）测定培养后成骨细胞纯度.采用FCM分别检测成骨细胞膜CCR1的表达水平,并在培养体系中加入CCL3干预后比较成骨细胞数量、形态及钙质沉积的变化.结果 经联合诱导培养获得的细胞形态不规则,有较多突起,碱性磷酸酶染色及Von Kossa＇s钙染色均为阳性,提示培养获得的细胞为成骨细胞,经FCM测定纯度均在90％以上.MM患者成骨细胞膜CCR1的表达水平为（74.48±7.31）％,高于正常对照组[（48.35±8.81）％]（P＜0.05）.加入CCL3干预后,MM患者成骨细胞形态及数量变化不显著,Von Kossa＇s钙染示钙结节明显减少.结论 采用骨髓来源的单个核细胞可用于体外培养MM患者成骨细胞.MM患者成骨细胞膜高表达CCR1,CCL3可抑制成骨细胞功能.提示CCL3可能在MM骨病成骨受抑机制中发挥一定作用.

Objective To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma （MM）.Methods Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone,β-sodium glycerophosphate and vitamin C,which were identified by alkaline phosphatase staining,Von Kossa＇ s staining.The CCL3 receptor expression was evaluated by flow cytometry.The morphology and quantity of osteoblast were observed after exposure to CCL3.Results Bone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa＇ s.MM-derived osteoblasts expressed higher levels of CCR1 （74.48±7.31）％,compared with normal controls （48.35±8.81）％.Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls.Conclusions Bone marrow osteoblasts could be cultured in vitro from MM Patients.CCL3 may contribute to the development of myeloma bone disease.