骨桥蛋白对骨髓间充质干细胞核力学特性的影响及相关分子机制

目的 研究骨桥蛋白（osteopontin, OPN）对骨髓间充质干细胞（bone marrow-derived mesenchymal stem cells, BMSCs）核力学特性的影响及相关分子机制。方法 采用Transwell法分析BMSCs迁移能力。利用原子力显微镜（atomic force microscope, AFM）检测细胞核弹性模量，分析OPN作用下BMSCs细胞核硬度的变化。通过Western blot技术检测OPN作用对黏着斑激酶（focal adhesion kinase, FAK）和胞外信号调节激酶1/2（extracellular signal-regulated kinase 1/2, ERK 1/2）的影响，并利用FAK或ERK1/2抑制剂考察FAK-ERK1/2信号通路在OPN影响BMSCs核力学特性中的作用。通过RT-PCR和Western blot技术检测OPN作用下核纤层蛋白Lamin A/C的表达变化。结果 OPN处理组细胞核弹性模量与对照组相比明显降低。OPN作用显著上调FAK、ERK1/2磷酸化水平，加入FAK或ERK1/2抑制剂在一定程度上回救OPN降低的细胞核弹性模量，并显著抑制BMSCs迁移。OPN处理BMSCs后Lamin A/C mRNA和蛋白水平的表达出现下调，但FAK或ERK1/2抑制剂能够抑制OPN诱导的Lamin A/C表达下调。结论 OPN可能通过FAK-ERK1/2信号通路下调BMSCs核骨架蛋白Lamin A/C表达，降低细胞核硬度，促进BMSCs迁移。该研究结果为深入认识OPN调控BMSCs迁移行为的机制及其临床应用提供了实验依据。
Objective To study the effects of osteopontin (OPN) on the nuclear mechanics of bone marrow-derived mesenchymal stem cells (BMSCs) as well as its involved mechanisms. Methods The BMSC migration was evaluated using the Transwell assay. An atomic force microscope (AFM) was used to determine the elastic modulus of the BMSC nucleus and analyze the changes in the nuclear mechanics of the BMSCs after treatment with OPN. The activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase1/2 (ERK1/2) was measured by Western blot. The role of the FAK-ERK1/2 signaling pathway in mediating the OPN-affected BMSC nuclear mechanics was investigated by employing a specific inhibitor. RT-PCR and Western blot were used to detect the expression of Lamin A/C at mRNA and protein levels in the BMSCs, respectively. Results The elastic modulus of the BMSC nucleus exhibited a significant decrease after OPN treatment compared with that of the control group. OPN could upregulate the phosphorylation level of FAK and ERK1/2, but the inhibitor of FAK or ERK1/2 restored the OPN-decreased elastic modulus of the BMSC nucleus and inhibited the BMSC migration significantly. After treatment with OPN, the expression of Lamin A/C in the BMSCs reduced significantly, and such a reduced expression could be suppressed by the inhibitor of FAK or ERK1/2. Conclusions OPN could probably downregulate the expression of Lamin A/C of the BMSCs via the FAK-ERK1/2 signaling pathway, decrease the stiffness of the BMSC nucleus, and promote the migration of the BMSCs. The research outcomes provide the experimental evidence for further understanding the mechanism of the OPN-regulated BMSC migration and its potential clinical application.