骨髓脂肪细胞促进多发性骨髓瘤细胞生存并上调抗药性

Bone Marrow Adipocytes Promote the Survival of Multiple Myeloma Cells and Up-Regulate Their Chemoresistance

目的:探讨多发性骨髓瘤(MM)患者骨髓微环境中脂肪细胞(Ad)在MM发生、发展中的作用。方法:油红O染色分析正常供者(HD)及初诊MM(ND-MM)患者骨髓涂片中骨髓脂肪细胞(BMA)含量。分别收集正常供者及初诊MM患者间充质干细胞(MSC),采用体外共培养试验探讨MM细胞对MSC成脂分化的影响以及BMA在MM细胞生存和耐药中的作用。实时定量PCR法检测MSC及MSC衍生Ad中成脂及成骨分化相关基因PPAR-γ、DLK1、DGAT1、FABP4、FASN和ALP表达,蛋白印迹法检测含或不含PPAR-γ抑制剂G3335共培养上清中IL-6、IL-10、SDF-1α、TNF-α和IGF-1水平。结果:油红O染色结果显示,与正常对照相比,ND-MM骨髓涂片阳性BMA显著增多,且与疾病状态相关,治疗有效者骨髓BMA含量下降;MM细胞明显上调了MSC成脂分化相关基因PPAR-γ、DLK1、DGAT1、FABP4和FASN的表达水平,而成骨分化相关基因ALP则明显下调。在MM骨髓微环境中MM细胞与MSC相互作用的直接后果是以成骨细胞为代价促进MSC向Ad分化,且该类Ad的细胞因子分泌谱发生变化。PPAR-γ抑制剂G3335可部分逆转BMA释放细胞因子,由此证实BMA经PPAR-γ信号调控其细胞因子释放,而G3335则可破坏PPAR-γ介导的BMA成熟和细胞因子释放。显著增多的BMA及相关细胞因子有效增强了MM细胞的增殖、迁移和耐药性。结论:BMA及其相关细胞因子是MM细胞生存、增殖和迁移的促进因素。MM衍生的BMA能保护MM细胞免受药物诱导的细胞凋亡,在MM治疗失败和疾病进展中起着重要作用。

Objective:To investigate the effect of adipocytes in the bone marrow microenvironment of patients with multiple myeloma (MM) on the pathogenesis of MM.Methods:Bone marrow adipocytes (BMA) in bone marrow smears of health donors (HD) and newly diagnosed MM (ND-MM) patients were evaluated with oil red O staining.The mesenchymal stem cells (MSC) from HD and ND-MM patients were isolated,and in vitro co-culture assay was used to explore the effects of MM cells on the adipogenic differentiation of MSC and the role of BMA in the survival and drug resistance of MM cells.The expression of adipogenic/osteogenic differentiation-related genes PPAR-γ,DLK1,DGAT1,FABP4,FASN and ALP both in MSC and MSC-derived adipocytes was determined with real-time quantitative PCR.The Western blot was employed to detect the expression levels of IL-6,IL-10,SDF-1α,TNF-α and IGF-1 in the supernatant with or without PPAR-γ inhibitor.Results:The results of oil red O staining of bone marrow smears showed that BMA increased significantly in patients of ND-MM compared with the normal control group,and the BMA content was related to the disease status.The content of BMA decreased in the patients with effective chemotherapy.MM cells up-regulated the expression of MSC adipogenic differentiation-related genes PPAR-γ,DLK1,DGAT1,FABP4 and FASN,but the expression of osteogenic differentiation-related gene ALP was significantly down-regulated.This means that the direct consequence of the interaction between MM cells and MSC in the bone marrow microenvironment is to promote the differentiation of MSC into adipocytes at the expense of osteoblasts,and the cytokines detected in supernatant changed.PPAR-γ inhibitor G3335 could partially reverse the release of cytokines by BMA.Those results confirmed that BMA regulated the release of cytokines via PPAR-γ signal,and PPAR-γ inhibitor G3335 could distort PPAR-γ mediated BMA maturation and cytokines release.The increased BMA and related cytokines effectively promoted the proliferation,migration and drug resistance of MM cells.Conclusion:The BMA and its associated cytokines are the promoting factors in the survival,proliferation and migration of MM cells.BMA can protect MM cells from drug-induced apoptosis and plays an important role in MM treatment failure and disease progression.