氧化型低密度脂蛋白对骨髓源性平滑肌样细胞增殖的影响

Effect of oxidative low-density lipoprotein on the proliferation of bone marrow stem cell-derived smooth muscle cells

目的建立体外诱导骨髓间质干细胞(BMSC)分化的平滑肌细胞模型,探索BMSC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BMSC,运用条件培养基诱导BMSC分化成SMC。采用免疫荧光法分析BMSC及其分化后的细胞特异性标志物。同时从胸主动脉分离血管平滑肌细胞(VSMC)作为对照。80mg/Lox-LDL分别处理两种细胞72h,细胞计数法观察这两种细胞的生长特征,流式细胞术分析细胞生长周期特征。结果 BMSC-SMC能大量蓄积胆固醇酯,形成泡沫细胞,这一表现较主动脉中膜分离而来VSMC更为明显。同时,BMSC-SMC和VSMC细胞的生长曲线大致呈S型,两种细胞的倍增时间分别约为20h和32h。经80mg/Lox-LDL处理后,BMSC-SMC和VSMC细胞的倍增时间分别约为15h和28h。结论在ox-LDL作用下,骨髓干细胞来源的平滑肌细胞增殖明显加快,并形成平滑肌源性泡沫细胞,提示BMSC-SMC可能参与脂质核心的形成,促进动脉粥样硬化的形成。

Objective To establish the model of bone mesenchymal stem cell-derived smooth muscle cells （BMSC-SMCs） and investigate the role of BMSC-SMCs in the development and progression of artherosclerosis. Methods BMSCs were isolated from the femoral bone of SD rats by adherent tissue culture method, and vascular smooth muscle cells （VSMCs） were obtained from the thoracic aorta. The differentiation of BMSCs into BMSC-SMCs was induced in the conditioned medium. The specific markers of BMSCs and BMSC-SMCs were identified by immunofluorescence （IF） staining. After treatment with 80 mg/L oxidative low-density lipoprotein （ox-LDL） for 72 h, the growth characteristics of BMSC-SMCs and VSMCs were observed. Flow cytometry was applied to analyze the cell cycle of BMSC-SMCs and VSMCs. Results BMCS-SMCs transformed into foam cells after treatment with ox-LDL, which was more obvious in comparison with VSMCs. The growth curve of BMSC-SMCs and VSMCs presented with an S-shape pattern with the cell doubling time of 20 and 32 h, which was reduced to 15 and 28 h after treatment with 80 mg/L ox-LDL, respectively. Flow cytometry showed that exposure to 80 mg/L ox-LDL significantly increased G0/G1 and decreased S and G2/M phase cells in both BMSC-SMCs （P0.01, n=3） and VSMCs （P0.05, n=3） in comparison with the control cells. Conclusion BMSC-SMC might be involved in the formation of fatty core and accelerate the development of atherosclerosis.