加热对血管平滑肌细胞的影响实验研究

Effects of Heating on Vascular Smooth Muscle Cells

为支架磁感应热疗预防和治疗PTCA术后血管再狭窄提供基础研究数据,对临床常用的316L型不锈钢冠脉支架进行磁感应诱导升温,探讨加热对兔血管平滑肌细胞(VSMCs)的增殖、迁移、凋亡及周期的影响。将平滑肌细胞置于恒温水浴槽中,待其达到预定温度(43、47℃)后持续10min,观察细胞形态变化,采用MTT法检测加热对血管平滑肌细胞增殖活性的影响,流式细胞术检测VSMC细胞周期和细胞凋亡,划痕实验检测加热对细胞迁移能力的影响,免疫细胞化学法检测不同温度作用后血管平滑肌细胞中增殖细胞核抗原(PCNA)的表达。结果发现,支架在交变磁场下可以升温至热疗所需温度,加热后细胞增殖受到了显著抑制,43℃组细胞存活率为(83.23±2.87)%,47℃组细胞存活率仅为(37.58±0.78)%。细胞的凋亡率显著增加,47℃组细胞凋亡率为(87.37±2.95)%,与对照组相比具有极显著差异,而43℃组的凋亡率为(6.00±0.26)%,与对照组相比无统计学差异。细胞周期也受到抑制,被阻滞在S期。加热后随着时间的推移,47℃组细胞的迁移能力受到显著抑制,而加热对43℃组细胞的迁移能力无显著影响。免疫细胞化学检测显示,随着温度的升高,PCNA的表达受到明显抑制。研究表明,临床常用冠脉支架在交变磁场下升温可行。加热显著抑制了细胞的增殖并促进了细胞凋亡,使细胞周期阻滞在S期,且抑制了细胞的迁移。加热能显著抑制细胞PCNA的表达,这些可能是加热抑制细胞增殖和迁移、促进凋亡的机制之一。

The aim of this study is to provide some basic research data for magnetic stent hyperthermia for restenosis prevention and treatment after PTCA induced heat of the 316L stainless steel stent with the Alternating Magnetic Field（AMF）.The effects of heating on the proliferation,migration,apoptosis and cell cycle of rabbit vascular smooth muscle cells are also studied.VSMCs were incubated at 43℃,47℃ or 37℃（control） in temperature-controlled water baths.The cell viability,apoptosis rate,cell cycle rate,cell migration and expression of PCNA protein were measured by MTT assay,flow cytometry,scratch assay and immunocytochemistry,respectively,after the therapy.Results show that heating of coronary stent could be induced by the alternating magnetic field.The proliferation of VSMCs was significantly inhibited by hyperthermia.The cell viability of the 43℃ group was（83.23±2.87）%,while that of the 47℃ group was only（37.58± 0.78）%.The apoptosis rate of the 47℃ group（87.37±2.95）% was significantly higher than that of the control group,while that of the 43℃ group saw no statistically significant difference compared with that of the control group.The cell cycle was arrested in S phase.After hyperthermia,the cell migration of the 47℃ group was significantly suppressed,but it had no influence on the cells of the 43℃ group.The expression of PCNA was decreased with the temperature of the bath.All above results suggest that the coronary stents can be heated by the alternating magnetic field.Hyperthermia can depress cell viability and induce VSMCs cell apoptosis.Also it can arrest the cell cycle in S phase and suppress the VSMCs cell migration.The expression of PCNA was reduced by hyperthermia,which may serve as the mechanism of the inhibition of hyperthermia on the proliferation and migration of VSMCs,and the promotion of VSMCs apoptosis.