香烟烟雾提取物对小鼠血管平滑肌细胞的损伤作用及表型转变的影响

Injury Effect and Phenotype Change on Mouse Vascular Smooth Muscle Cells Induced by Cigarette Smoke Extract

目的探讨香烟烟雾提取物(CSE)对小鼠血管平滑肌细胞的损伤作用及表型转变的影响。方法制备CSE,分别使用2.5%、5%、10%、20%CSE处理小鼠血管平滑肌细胞(MOVAS细胞),并以正常MOVAS细胞作为对照组。MTT法检测各浓度CSE处理后细胞的存活率,活性氧(ROS)检测试剂盒检测细胞内ROS水平。流式细胞仪结合PI单染以及Annexin V-PI双染技术分别分析10%CSE处理后细胞周期和细胞凋亡率的变化。实时荧光定量PCR分析10%CSE处理后MOVAS细胞内SMemb和SMA的mRNA表达水平,Western blot检测10%CSE处理后MOVAS细胞内SMemb和SMA的蛋白表达水平。结果 2.5%、5%、10%及20%CSE处理后,MOVAS细胞的存活率分别为89.6%±8.6%、65.2%±10.0%、53.6%±6.9%、43.1%±5.9%,各组与对照组相比差异具有统计学意义(P<0.05);各浓度CSE处理后细胞内ROS水平显著提高。10%CSE处理后细胞G1期比例明显上升,细胞凋亡率也显著提高,与对照组相比差异具有统计学意义(P<0.05)。10%CSE处理后MOVAS合成型标志物SMemb的表达水平显著增高,而收缩型标志物SMA的表达水平显著降低。结论 CSE可以对小鼠血管平滑肌细胞造成损伤,并能够使小鼠血管平滑肌细胞表型由收缩型向合成型转变,其机制可能与CSE引起的细胞内ROS增高有关。

Aim To explore injury action and phenotype shift on mouse vascular smooth muscle cells by cigarette smoke extract( CSE). Methods CSE was prepared,2. 5%,5%,10% and 5% of CSE was respectively used to treat mouse vascular smooth muscle cells( MOVAS cells),and normal MOVAS cells were used as control group. Cell survival rate was detected by MTT assay. Intracellular reactive oxidative species( ROS) level was estimated by ROS kit.MOVAS cell cycle and apoptotic rate were respectively analyzed by flow cytometry instrument combining PI simple staining and Annexin V-PI double staining. SMemb and SMA mRNA expression of MOVAS cells were analyzed by real-time quantitative PCR,SMemb and SMA protein expression of MOVAS cells were measured by Western blot. Results After2. 5%,5%,10% and 20% CSE processed,survival rate of MOVAS cells was respectively 89. 6% ± 8. 6%,65. 2% ±10. 0%,53. 6% ± 6. 9% and 43. 1% ± 5. 9%,the difference was statistically significant compared with the control group( P < 0. 05),meanwhile,ROS level increased significantly. After 10% CSE processed,the percent of cells in G1 cycle was increased obviously,the difference was statistically significant( P < 0. 05). Cell apoptosis rate also increased significantly,the difference was statistically significant( P < 0. 05). The expression of SMemb as MOVAS synthetic symbol was significantly increased,while the expression of SMA as contractile symbol was reduced remarkably. Conclusion CSE can injure mouse vascular smooth muscle cells,and promote the transformation of MOVAS cells’ phenotype from synthetic to contractile.