阿糖胞苷通过自噬途径影响K562细胞增殖凋亡的实验研究

Experimental study on influence of cytarabine on K562 cells proliferation and apoptosis by autophagy pathway

目的探讨阿糖胞苷(Ara-C)通过自噬途径影响人红白血病K562细胞株增殖、凋亡的作用及可能的机制。方法采用CCK-8法检测不同浓度的Ara-C作用24h和48h后细胞增殖抑制率;流式细胞术(FCM)检测凋亡率和周期;Hoechest染色观察细胞核染色质的形态,吖啶橙染色观察细胞酸性自噬小泡;Western blot检测p38和p-p38蛋白表达变化;RT-PCR和免疫荧光检测自噬凋亡相关基因和蛋白的表达水平。结果 CCK-8检测发现不同浓度的Ara-C均能抑制K562细胞增殖,并呈浓度和时间依赖性;FCM检测显示Ara-C能增加细胞的凋亡和将细胞周期阻滞在S期;Hoechest染色发现Ara-C处理K562细胞后呈凋亡形态改变;吖啶橙染色发现Ara-C组细胞绿色荧光增强,细胞出现大量的酸性自噬小泡;RT-PCR检测发现Ara-C上调自噬关键基因Beclin-1、LC3A和LC3B表达;Western blot检测发现Ara-C增加磷酸化p38表达;免疫荧光检测发现Ara-C增加LC3表达。结论 Ara-C能够激活p-p38介导的K562细胞发生自噬,进而抑制细胞增殖和促进细胞凋亡作用。

Objective To investigate the effect of cytarabine（Ara-C）on proliferation and apoptosis of human erythroleukemia K562 cell line through autophagy pathway and its possible mechanism.Methods The cellular proliferation inhibiting rate after different concentrations of Ara-C acting for 24,48 hwas detected by CCK-8;the cell cycle and apoptosis were detected by flow cytometry（FCM）;the chromatin morphological changes in nucleus were observed by Hoechst staining;the cell acidic autophagy vesicles were detected by acridine orange staining;the expression changes of p38 and p-p38 proteins were detected by Western blot.The expressions of autophagy apoptosis related gene and protein were examined by RT-PCR and immunofluorescence.Results The CCK-8results found that different concentrations of Ara-C could inhibit the proliferation of K562 cells with dose-and time-dependent manners.FCM detecting indicated that Ara-C could increase apoptosis and could arrest the cell cycle at S phase;Hoechest staining showed that K562 cells had typical apoptotic morphological changes after Ara-C treating;the Acridine orange staining revealed that Ara-C caused the inclease of the green fluorescene in cells of the Ara-C group,and the cells appeared a great number of acidic autophagy vesicles;RT-PCR results showed that Ara-C up-regulated the expression of autophagy key genes Beclin-1,LC3 Aand LC3B;Western blot results showed that Ara-C increased the expression of phosphorylated p-p38.Immunofluorescence results showed the expression of LC3 Bwas significantly enhanced.Conclusion Ara-C can activate p-p38 mediated K562cells to generate autophagy,then inhibit the cell proliferation and promotes apoptosis.