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

作　　者：罗昊[1] 孟赞[1] 刘泽洪 陈晓露[3] Luo Hao Meng Zan Liu Zehong Chen Xiaolu(Department of Human Anatomy, Histology and Embryology, Leshan Vocational and Technical College, Leshan, Sichuan 614000, China Department of Pathology, Three Gorges Medical College, Chongqing 404120, China Department of Pathology ,Leshan Vocational and Technical College ,Leshan, Sichuan 614000 ,China)

机构地区：[1]乐山职业技术学院人体解剖学与组织胚胎学教研室,四川乐山614000 [2]三峡医药高等专科学校病理学教研室,重庆万州404120 [3]乐山职业技术学院病理学教研室,四川乐山614000

出　　处：《重庆医学》2017年第13期1736-1739,共4页Chongqing medicine

摘　　要：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.

关 键 词：阿糖胞苷 白血病 自噬 细胞凋亡 

分 类 号：R96[医药卫生—药理学]