氧化型低密度脂蛋白对骨髓源性平滑肌样细胞粘附和迁移的影响

Effect of ox-LDL on Adhesion and Migration in Smooth Muscle Cell Derived from Bone Marrow Stem Cell

【目的】建立体外诱导骨髓间质干细胞分化的平滑肌细胞模型,探索骨髓间质干细胞分化的平滑肌细胞在动脉粥样硬化发生发展过程中的作用。【方法】采用贴壁法从SD大鼠骨髓中分离骨髓间质干细胞(BMSC),运用条件培养基诱导BMSC分化成平滑肌细胞(SMC),采用免疫荧光法分析骨髓间质干细胞及其分化后的细胞特异性标志物。羟脯氨酸测定法检测两种细胞合成细胞外基质能力,粘附和迁移实验检测两种细胞的粘附和迁移能力。【结果】①分离的BMSC呈长梭形,呈骨髓间质干细胞表型。经条件培养基诱导分化15d后,诱导分化的细胞形态呈梭形平滑肌样。表达平滑肌细胞特异性蛋白标志物α-肌动蛋白(α-SMC)和平滑肌肌球蛋白重链1(SM-MHC1),呈平滑肌细胞表型,命名为骨髓间质干细胞分化的平滑肌细胞(BMSC-SMC)。②羟脯氨酸测定结果显示,未处理BMSC-SMC合成较高量的胶原,其合成量低于VSMC的合成量,经80mg/Lox-LDL处理72h后,BMSC-SMC合成胶原的能力显著降低(P<0.01)。③细胞粘附实验结果发现,未处理BMSC-SMC有中等数量的细胞发生粘附,经80mg/Lox-LDL处理1h后,BMSC-SMC细胞粘附的数目少量增加,但两者比较差异无统计学意义(P>0.05);经80mg/Lox-LDL处理24h后,BMSC-SMC细胞迁移的数目显著增多,与未处理BMSC-SMC比较,差异具有统计学意义(P<0.01)。【结论】①经条件培养基诱导培养后,骨髓干细胞表达平滑肌特异性标志物,SMα-SMC和SM-MHC1,提示骨髓干细胞能成功分化成平滑肌细胞。②骨髓干细胞诱导成平滑肌细胞后,具有血管平滑肌细胞的一些生物学特征,如增殖能力和分泌胞外基质能力,同时保留了骨髓干细胞的一些生物学特征,如高迁移能力,为其在促动脉粥样硬化因素作用下,跨内皮细胞层和增殖的平滑肌细胞层迁移创造了有利的条件。

【Objective】To establish the model of smooth muscle cell differentiated from bone mesenchymal stem cell（BMSC-SMC）in vitro and investigate the roles of BMSC-SMC in the occurrence and development of artherosclerosis（As）.【Methods】 BMSC was isolated from the femoral bone of SD rats by adherent method,and VSMC from thoracic aorta.BMSC-SMC was differentiated from BMSC by special condition medium.The specific markers of BMSC and smooth muscle cell differentiated from bone mesenchymal stem cell（BMSC-SMC）were identified by immunofluorescence（IF）staining.After treated with 80 mg/L oxLDL for 72 h,hydroxyproline assay was performed to detect the extracellular matrix synthesis capacity,and adhesion and migration experiments were used to assay the adhesive and migratory capacity of BMSC-SMC and VSMC.【Results】①The isolated BMSC present a slim-spindle appearance.The cells（BMSC-SMC）induced by condition medium containing b-FGF and TGF-βfor15 days present slim-spindle smooth muscle-like cells.Immunofluorescence（IF）staining showed that the cells expressed smooth muscleα-actin and smooth muscle myosin heavy chain l,which were the specific markers of SMC.The cells are named smooth muscle cell differentiated from bone mesenchymal stem cell（BMSC-SMC）.②Hydroxyproline assay showed that the amount of collagen synthesis was high in control BMSC-SMC,which was lower than that of VSMC.After treated with 80 mg/L ox-LDL for 72 h,the ability of collagen synthesis was significantly decreased in BMSC-SMC,and was also obviously decreased in VSMC.The results of cell adhesion experiment showed that the amounts of cell adhesion were medium in control BMSC-SMC,which was lower than that of VSMC.After treated with 80 mg/L ox-LDL for 1 h,the amounts of cell adhesion were rarely increased in BMSC-SMC,and were rarely decreased in VSMC.The difference was not significant compared with the control respectively.The Cell damagerepair experiment showed that the amount and distance of migration were significantly increased in BMSC-SMC compared with that of in control BMSC-SMC（P0.01）.【Conclusion】①BMSC expressed the specific markers of SMC,such as SMα-SMC and SM-MHC1,which indicated that BMSC had differentiated into SMC.②BMSC-SMC have some biological characteristics that similar with VSMC,such as the ability of proliferation and secretion of extracellular matrix,but at the same time retain some biological characteristics of BMSC,such as the ability of high migration,which provides a favorable condition for its migration from endothelial cell layer and hyperplastic smooth muscle cell layer to atherosclerotic plaque layer under promote atherosclerosis factors.