平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其机制的实验研究

目的：探讨平阳霉素对人脐静脉内皮细胞系ECV304的抑制作用及其相关机制。方法：用不同浓度的平阳霉素作用ECV304细胞不同时间，以MTT法比较平阳霉素作用24、48及72h后的细胞抑制率，应用DNA电泳实验、流式细胞术分析细胞凋亡、坏死情况、细胞周期及Fas、Bcl-2蛋白的表达。结果：MTT法显示随平阳霉素浓度升高和作用时间的延长，细胞生长抑制越明显DNA电泳证实合适浓度平阳霉素作用下细胞发生凋亡，过高浓度细胞则发生坏死；流式细胞术显示细胞凋亡率和坏死率随药物浓度和作用时间增加而增高，过高浓度细胞坏死占主导；平阳霉素各浓度作用24h后，G2—M期百分率增高，S期百分率降低，G0—G1期百分率无明显变化；Fas表达随药物浓度增加而递减，Bcl-2表达随药物浓度增加而升高。结论：平阳霉素能明显抑制ECV304细胞生长，并具有浓度和时间依赖性；平阳霉素可能通过诱导凋亡和坏死作用以抑制ECV304细胞的体外生长和增殖；细胞凋亡发生在G2—M期，是通过上调Fas蛋白并下调Bcl-2蛋白的表达来实现的。

Study on the Inhibitory Effect and Mechanism of Pingyangmycin Against ECV304 Cell

Objective:To investigate the inhibitory effect of Pingyangmycin(PYM) on cultured human umbilical vein endothelial cell line ECV304 in vitro and the possible mechanism.Methods: The inhibitory rates of ECV304 cells,which were treated with PYM for 24h,48h and 72h,were compared respectively by MTT assay.The changes of apoptosis and necrosis of the cells treated with PYM for 24h were analyzed by electrophoresis of DNA.The apoptotic rate,necrotic rate,cell cycle and protein expression of Fas and Bcl-2 were examined by flow cytometry(FCM).Results: ECV304 cells were inhibited more significantly with increasing PYM concentration and prolonging culture time.PYM with appropriate concentration may induce apoptosis in ECV304 cells,but higher concentration may lead necrosis of the cells.Apoptotic rate and necrotic rate increased with PYM concentration and culture time.Otherwise, necrosis prevailed over apoptosis at higher concentration.The percentage of G2-M phase cells increased while the S phase decreased,but the percentage of G0-G1 phase cells remained unchanged after treated with PYM for 24h.The expression of Fas increased with PYM concentration while the expression of Bcl-2 decreased.Conclusion: PYM can inhibit ECV304 cells in vitro effectively in a concentration-dependent and time-dependent manner.The mechanisms of PYM on growth inhibition of ECV304 cells in vitro may include apoptosis induction and necrosis effect.The apoptosis of ECV304 cells happens in the cell cycle of G2-M phase through the up-regulation of Fas expression and down-regulation of Bcl-2 expression.