电场对血管内皮细胞增殖及其细胞周期的影响

目的 观察电场对血管内皮细胞增殖的影响并探讨其作用机制。方法 将血管内皮细胞分别暴露于场强为50mVmm^-1,100mVmm^-1和200mVmm^-1的电场7。部分细胞接受细胞凋亡抑制剂Z－VAD－FMK的同时暴露于200mVmm^-1的电场。计算细胞密度，生长率和分裂指数，行细胞凋亡信号蛋白caspase-3免疫荧光染色，以流式细胞仪行细胞周期时相分析和通过蛋白印记分析检测细胞周期调节蛋白的表达。结果 50mVmm^-1或100mVmm^-1的电场暴露对细胞增殖没有明显影响，但200mVmm^-1的电场细胞增殖明显受抑，表现为细胞密度，生长率及分裂指数明显下降。Z－VAD－FMK未能防止电场暴露所诱发的细胞增殖的抑制。电场暴露细胞未见有明显的caspase-3阳性染色。电场暴露细胞的细胞周期时相图上，亦未见提示细胞凋亡的亚G1峰，而显示其细胞周期的G1／S过渡受抑。电场暴露细胞的Cdk2表达不受影响，但Cyclin-E的表达明显降低，而cyclin-E/Cdk2复合物的抑制蛋白p27^kip1的表达则明显增加。结论 电场暴露通过诱发细胞周期停滞而非细胞凋亡抑制血管内皮细胞的增殖，该作用是籍增加p27^kip1的表达和减少cyclin-E的表达而实现。

Electric field inhibits vascular endothelial cell proliferation by cell cycle control

Objective To observe the effects of electric fields (EF) on proliferation of vascular endothelial cells (VEC) and probe the underlying mechanisms. Methods VEC were exposed to EF at 50,100 or 200 mV mm -1 for up to 72 hours, and some cells incubated with apoptosis inhibitor Z VAD FMK were exposed to EF of 200 mV mm -1 . Cell density, cell growth rate and mitosis index were measured. Immunofluorescence staining for active caspase-3 was performed in VEC. Flow cytometry was used for the analyses of cell cycle phases and Western blotting for the analyses of expression of cyclin E, Cdk2 and p27 kip1 . Results EF at 50 or 100 mV mm -1 did not influence VEC proliferation, but EF at 200 mV mm -1 inhibited ceu proliferation, with significant decreases of cell density, cell growth rate and mitosis index. EF induced reduction in VEC proliferation was not prevented by Z VAD FMK. VEC exposed to EF of 200 mV mm -1 did not show significant active caspase 3 staining and with flow cytometry, a sub-G1 peak indicating apoptosis was not observed on the DNA histogram of EF exposed cells. EF decreased the entry of cells into S phase from G1 phase, inhibiting the transition of G1/S phases. Also EF decreased the expression of cyclin E, a G1 specific cell cycle protein and increased the expression of the cyclin Cdk complex inhibitor p27 kip1 . Conclusion EF inhibit VEC proliferation by cell cycle arrest rather than apoptosis, which is mediated by down regulation of cyclin E expression and up regulation of p27 kip1 expression.