骨髓瘤细胞抑制骨髓间充质干细胞向成骨细胞的转化

Multiple myeloma cells inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells

目的:多发性骨髓瘤患者的长期生存率没有得到提高,因此进一步明确多发性骨髓瘤的发病机制并寻找理想的治疗方法是当前研究的重要课题。实验观察多发性骨髓瘤细胞对骨髓间充质干细胞成骨分化的潜能及对其表达核因子κB受体激活剂配体/骨保护蛋白的影响。方法:选择2006-07/2007-06在苏州大学附属第二医院住院的7例多发性骨髓瘤患者,患者经骨髓细胞学检查、血液生化测定及免疫球蛋白定量分析等符合多发性骨髓瘤诊断标准。正常成人骨髓由苏州大学附属第二医院血液科研究生捐献。患者对实验知情同意,并经医院伦理委员会批准。实验方法:常规分离培养两种来源骨髓间充质干细胞,采用直接免疫荧光标记及流式细胞术分析其表面标志。观察两种来源骨髓间充质干细胞在成骨诱导培养条件下向成骨细胞分化的潜能,并在诱导培养的第1天和3周时分别加入RPMI8266或XG7多发性骨髓瘤细胞株,于培养28d后行Von-Kossa及TRAP染色;多发性骨髓瘤细胞与正常骨髓间充质干细胞共培养24h后,应用RT-PCR法检测核因子κB受体激活剂配体/骨保护蛋白的表达。结果:①两种骨髓间充质干细胞形态无明显区别,表型特征相似,均可在成骨诱导培养后向成骨细胞分化。②Von-Kossa染色后提示来源于多发性骨髓瘤患者的骨髓间充质干细胞成骨分化潜能较差,矿化基质沉积较正常来源骨髓间充质干细胞少。两种骨髓间充质干细胞共培养第28天行Von-Kossa染色后,可见矿化基质沉积较不加入多发性骨髓瘤细胞株组明显减少(P<0.001),TRAP染色未发现破骨样细胞的存在。③RT-PCR结果显示,正常骨髓间充质干细胞不表达核因子κB受体激活剂配体,但表达骨保护蛋白,与骨髓瘤细胞共培养后,8226或XG7细胞可明显上调骨髓间充质干细胞表达核因子κB受体激活剂配体,相反骨保护蛋白表达明显受抑。结论:多发性骨髓瘤细胞抑制骨髓间充质干细胞的成骨分化及上调骨髓间充质干细胞表达核因子κB受体激活剂配体,抑制骨保护蛋白的表达可能是多发性骨髓瘤患者骨病发病的主要原因。

AIM： The long-time survival rate of multiple myeloma （MM） is still poor now. So, to identify the pathogenesis and search a good therapy seems very important. In this study, the effects of MM cells on osteogenic differentiation of bone marrow mesenchymal stem cells （MSCs） and the expression of receptor activator of nuclear factor-κB ligand （RANKL） /osteoprotegerin （OPG） were investigated. METHODS： Seven MM inpatients were selected from Second Hospital of Suzhou University from July 2006 to June 2007. They were diagnosed according to the bone marrow cytomorphologic examination, blood biochemical detection and quantitative analysis of immune globulin （IG）. Meanwhile, the bone marrow was donated by the postgraduates of Department of Hematology, Second Hospital of Suzhou University. The experiment was agreed by the patients and permitted by the Hospital Ethics Committee. MSCs from bone marrow of MM patients and normal persons were separated and cultured. Direct immunofluorescence assay was employed to analyze the phenotype of those cells by using flow cytometry. The osteogenic potential of those two MSCs was investigated under osteogenic condition medium with or without MM cell line RPMI8226 or XG-7 in vitro at the first day and the third week. Von-Kossa and TRAP staining was performed after the MSCs were incubated with MM cells for 28 days. RT-PCR assays were used to detect the expression of RANKL/OPG of MSCs after 24 hours co-culture with MM cells. RESULTS： ①The growth character and morphological pattern of the cells observed under light microscope were nearly the same. Both MSCs could differentiate towards osteoblast under osteogenic induction condition. ②Von-Kossa staining showed that the MSCs from MM patients presented poor osteogenic differentiation with mineral deposition in osteogenic condition medium. In co-culture groups, mineral deposition was decreased significantly in those cells incubated with MM cells （P 〈 0.001 ） at the 28^th day after Von-Kossa staining. By using TRAP staining, no osteoclast-like cell was found. ③ Normal MSCs did not express RANKL but OPG, while after co-cultured with MM cells, 8226 or XG7 cells could significantly upregulate the RANKL expression and downregulate the OPG expression of MSCs as measured by RT-PCR assay. CONCLUSION： expression and myeloma. Multiple myeloma cells inhibit osteogenic differentiation potential of MSCs, upregulate downregulate the OPG of MSCs, which may be one of main causes of the occurrence of RANKL multiple myeloma.