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目的:研究凋亡调控相关蛋白来了解顺铂耐药成因,同时考察乙烷硒啉(Ethaselen)在K562耐药细胞中逆转顺铂耐药的作用,并初步探讨其作用机制。创新点:首次研究乙烷硒啉在逆转顺铂耐药中的作用,且此作用与乙烷硒啉诱导细胞凋亡相关。方法:通过长时间脉冲诱导得到顺铂耐药K562细胞,并观察耐药细胞形态及倍增时间。采用MTT法考察乙烷硒啉、顺铂及其联用组在不同细胞株间的生长抑制作用。流式细胞术分析细胞凋亡情况以及细胞内活性氧(ROS)水平。最后,通过蛋白质免疫印迹(Western blot)考察凋亡调控相关蛋白水平的变化。结论:脉冲诱导得到的K562耐药细胞对顺铂的耐受性是原K562细胞的5.34倍。形态学观察发现,耐药细胞体积增大,粘附性进一步降低。乙烷硒啉与顺铂联用表现出协同效应。当加入少量的乙烷硒啉(顺铂与乙烷硒啉的摩尔比率为10:1),顺铂作用K562耐药细胞的半抑制浓度(IC50)值可以减少21倍。流式细胞术及Western blot表明,乙烷硒啉能够诱导耐药细胞凋亡。其逆转顺铂耐药主要是通过调控Bcl-2及Bax蛋白比例以及通过提高细胞内活性氧水平引起线粒体通透转运孔道(PTP)蛋白孔道的形成来促使释放细胞色素c,进而引起Caspase凋亡途径。
OBJECTIVE: To study the genesis of cisplatin resistance in the study of apoptosis related proteins, and to investigate the role of ethaselen in reversing cisplatin resistance in K562 resistant cells and to explore its mechanism. Innovation: The first study of ethaselen in reversing the role of cisplatin resistance, and this role and ethaneselen-induced apoptosis. Methods: C5-resistant K562 cells were induced by long-term pulse and the morphology and doubling time of drug-resistant cells were observed. MTT assay was used to investigate the growth inhibitory effect of ethaselen, cisplatin and their combination on different cell lines. Flow cytometry analysis of apoptosis and intracellular reactive oxygen species (ROS) levels. Finally, Western blotting was used to investigate the changes of apoptosis related proteins. Conclusion: The resistance of K562 resistant cells to cisplatin induced by pulse is 5.34 times of that of K562 cells. Morphological observation showed that drug-resistant cells increased in size and adhesion decreased further. Ethaselenol in combination with cisplatin showed a synergistic effect. When a small amount of ethaselin was added (the molar ratio of cisplatin to ethaselenol was 10: 1), the half-inhibitory concentration (IC50) of cisplatin-resistant K562-resistant cells could be reduced by 21-fold. Flow cytometry and Western blot showed that ethaselen can induce drug-resistant cell apoptosis. Its reversal of cisplatin resistance mainly through the regulation of Bcl-2 and Bax protein ratio and through the increase of intracellular levels of reactive oxygen species caused by the formation of mitochondrial PTR protein pore to promote the release of cytochrome c, and then cause apoptosis of Caspase way.