罗 昊，孟 赞，刘泽洪，陈晓露

(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)

论著·基础研究

阿糖胞苷通过自噬途径影响K562细胞增殖凋亡的实验研究

罗 昊1，孟 赞1，刘泽洪2，陈晓露3△

(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)

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24 h和48 h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。

阿糖胞苷；白血病；自噬；细胞凋亡

[Abstract] 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 h was 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-8 results 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 K562cells 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,LC3A and LC3B;Western blot results showed that Ara-C increased the expression of phosphorylated p-p38.Immunofluorescence results showed the expression of LC3B was significantly enhanced.Conclusion Ara-C can activate p-p38 mediated K562 cells to generate autophagy,then inhibit the cell proliferation and promotes apoptosis.

红白血病是以红、白(主要是粒细胞)两系恶性增生的白血病，其发病机制为造血干细胞分化障碍，细胞周期调控紊乱，细胞凋亡受阻的恶性造血系统肿瘤[1]。……