骨髓间充质干细胞Notch1信号通路异常与骨髓瘤骨病

Effect of Notch1 signaling abnormality on the pathogenesis of myeloma bone disease

背景:多发性骨髓瘤骨病的发病机制目前尚未完全明确,骨髓间充质干细胞向成骨细胞分化障碍参与其中,而Notch1信号通路在间充质干细胞的增殖分化中起重要作用。目的:探讨Notch1信号通路在多发性骨髓瘤骨病中的作用。方法:分离培养多发性骨髓瘤患者和正常人骨髓间充质干细胞,Real-time PCR和Western blot检测成骨诱导分化前后Notch1和成骨基因Runx2的表达,以及Von Kossa染色鉴定钙质沉积程度。在多发性骨髓瘤患者间充质干细胞成骨诱导分化过程中,加入Notch1信号通路抑制剂DAPT和安慰剂,48 h后real-time PCR和western blot鉴定Notch1信号通路下游分子Hes1和成骨指标Runx2表达,2周后Von Kossa染色鉴定钙质沉积程度。结果与结论:成骨诱导48 h后,间充质干细胞的Notch1表达减低,但是骨髓瘤患者间充质干细胞的降低幅度小于正常对照间充质干细胞;48 h后Runx2的表达在骨髓瘤患者间充质干细胞的表达明显弱于正常对照间充质干细胞;2周后,Von Kossa染色鉴定钙质沉积程度,骨髓瘤患者间充质干细胞明显弱于正常对照间充质干细胞;48 h后Hes1表达在DAPT组明显低于安慰剂组;而Runx2的表达在DAPT组明显高于安慰剂组。2周后DAPT组钙质沉积明显强于安慰剂组。实验说明多发性骨髓瘤患者的间充质干细胞中,Notch1信号通路失活缺陷可能抑制其向成骨细胞分化。

BACKGROUND： The pathogenesis of multiple myeloma bone disease is far from elucidated. The impaired osteogenic differentiation of bone marrow mesenchymal stem cells leads to osteoblast deficiency. Notch1 signaling has a key role in the proliferation and differentiation of bone marrow mesenchymal stem ceils. OBJECTIVE： To explore the role of Notch1 signaling in the pathogenesis of myeloma bone disease. METHODS： Bone marrow mesenchymal stem cells from myeloma patients and normal donors were isolated and cultured. The expressions of Notch1 and osteogenic marker Runx2 were detected with real-time PCR and westem blot before and after osteogenic induction. Von kossa staining was employed to detect calcium deposition. DAPT, an inhibitor of notch1 signaling, and placebo were added to the osteogenic medium of bone marrow mesenchymal stem cells, respectively. The expression of Hesl, a downstream gene of Notch1 signaling, and Runx2 were tested using real-time PCR and western blot after 48 hours. Von kossa staining was used to detect calcium deposition after 2 weeks.RESULTS AND CONCLUSION： After cultured in osteogenic medium for 48 hours, Notch1 expression of bone marrow mesenchymal stem cells from myeloma patients decreased, which was not as much as that of bone marrow mesenchymal stem coils from normal donors. This was accompanied by much more decreased expression of Runx2 and calcium deposition in the bone marrow mesenchymal stem coUs from myeloma patients compared with those from normal donors. Addition of DAPT inhibited Hesl and enhanced Runx2 expression after 48 hours, which was accompanied with more calcium deposition in bone marrow mesenchymal stem cells from myeloma patients after 2 weeks. These experimental data above suggest that the deactivation of Notch1 signaling is blocked, which is possibly related to the impaired osteogenic differentiation potential in the bone marrow mesenchymal stem ceils from myeloma patients.