汉防己甲素对小鼠血管内皮瘤细胞的影响及其分子机制研究

摘要

Objective To explore tetrandrine on murine hemangioendothelioma cell and its mechanisms.Methods Explore the effect of tetrandrine on EOMA cells in time and concentration MTT experiment, the content of tetrandrine treated EOMA cells in different cell cycle detected by flow cytometry, speculated effect of tetrandrine on G1/S phase checkpoint for EOMA cells blocking.Western blot analyzed the molecular mechanism of tetrandrine induced EOMA cell block, and intracellular reactive oxygen species level were detected byflow cytometry.Then used the active oxygen scavenger NAC pretreated cells after drug incubation.Results Tetrandrine could inhibit the cell reproduction, and showed a concentration and time dependence, role in tetrandrine 20μM was significantly inhibited after 48 h.Tetrandrine caused mainly by EOMA cells during the cell cycle of G1/S phase cell block, so as to achieve the effect of inhibiting cell proliferation.Tetrandrine increased intracellular reactive oxygen species ( ROS) blockade effect of elevated levels of cell cycle to achieve, and the regulation of AKT, GSK-3 βand p53 cell cycle upstream protein level, finally maked the expression changes.Further research proved that, tetrandrine induced up-regulation of ROS level in cells to induce EOMA cell cycle arrest.Moreover, regulated by the ROS inhibitor NAC could significantly inhibit EOMA cell cycle factor.Conclusion Tetrandrine can inhibit tumor angiogenesis, which provides theory basis for clinical anti-cancer drugs in inhibiting tumor growth.%目的：探求汉防己甲素对小鼠内皮瘤细胞系EOMA 细胞的影响及其分子机制。方法采用 MTT方法考察汉防己甲素在时间和浓度上对EOMA细胞的影响；使用流式细胞术检测汉防己甲素处理后的EOMA细胞内各个不同细胞周期内的含量，推测汉防己甲素对EOMA细胞的G1／S期检验点的影响；Western blot方法分析汉防己甲素引起EOMA细胞阻滞的分子机制，并用流式细胞术检测细胞内活性氧水平，用活性氧清除剂NAC预处理细胞后再进行药物孵育测定。结果汉防己甲素能够有效抑制此细胞的增殖，随着浓度增加时间延长，细胞增殖率下降；在汉防己甲素20μM 时作用48 h后便有显著的抑制作用。汉防己甲素对EOMA细胞周期中G1／S期的细胞阻滞和升高细胞内活性氧（ reactive oxygen species，ROS）水平、上调AKT， GSK－3β以及p53等上游蛋白水平，从而使细胞周期阻滞，使周期蛋白的表达发生变化。而ROS的抑制剂NAC可以明显地抑制EOMA细胞周期因子的调控。结论汉防己甲素可以抑制肿瘤血管新生，为后续研究将汉防己甲素作为临床抗癌药物提供了理论依据。