促炎因子介导的体外培养人主动脉血管平滑肌细胞凋亡

Apoptosis of Vascular Smooth Muscle Cells Induced by in Vitro Stimulation With Proinflammatory Cytokines

目的 :探讨促炎因子对体外培养的人主动脉平滑肌细胞有无致凋亡作用。 方法 :体外原代培养人主动脉平滑肌细胞 ,用免疫组化方法鉴定 ;肿瘤坏死因子 α (TNF α)和白细胞介素 1β (IL 1β)分别以浓度 2 0 μg/ml单独或二者共同与平滑肌细胞培养 3 6h ,实验分 4组 :TNF α组 (T组 )、IL 1β组 (I组 )、TNF α+IL 1β组 (TI组 )及对照组 ,采用流式细胞仪分析细胞凋亡及细胞周期。 结果 :①细胞凋亡分析显示 :TI组两种因子的联合作用诱导人平滑肌细胞凋亡与对照组比明显增加 ,有显著性差异 (P <0 0 5 ) ,而二者单独作用未能诱导人平滑肌细胞凋亡。②细胞周期分析发现TI组在TNF α +IL 1β刺激下 ,细胞增殖能力与其他组比有明显下降 ,有显著性差异 (P <0 0 5 ) ,生长逐渐停滞 ,而二者单独作用对细胞增殖有轻微刺激作用 ,但与对照组无显著性差异。 结论 :促炎因子TNF α和IL 1β的相互作用 ,可以诱导人平滑肌细胞凋亡。

Objective: To explore whether apoptosis of vascular smooth muscle cells (VSMCs) can be induced by in vitro stimulation with proinflammatory cytokines,tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β). Methods: Human VSMCs were isolated from the tunica media of aortas of 4month old embryo. The cells were cultured in Dulbecco's minimal essential medium supplemented with 10% fetal bovine serum and antibiotics. The cells were identified as VSMCs by immunofluorescence with anti-α-smooth muscle actin monoclonal antibody. To determine the effects of proinflammatory cytokines on the cultured VSMCs in vitro, the cells were exposed to TNF-α and interleukin-1β alone or together for 36 hours. After incubation with or without cytokines, the cells were suspended in fresh medium containing propidium iodine for 5 minutes at 37℃ and then subjected to a fluorescence-activated cell sorter flow cytometer immediately after incubation. Results: ①Apoptosis of VSMCs was triggered by in vitro stimulation with proinflammatory cytokines when simultaneous treatment with both TNF-α and interleukin-1β(p<0.05).However, the same result was not seen in the other groups(p>0.05). ②Analysis of cell cycle indicated that the ability of cell proliferation decreased more significantly in simultaneous treatment with both TNF-α and interleukin-1β than in the control group, meanwhile, the same result was not seen in the single cytokine-treated VSMCs groups(p<0.05). Conclusion: Cytokine-producing cells (eg, macrophages and T lymphocytes) abound in advanced atherosclerotic lesions, our present study suggested that proinflammatory cytokines may induce apoptosis of VSMCs.