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目的探讨西罗莫司对人急性早幼粒白血病细胞HL60增殖、自噬、凋亡的影响。方法四甲基偶氮唑蓝(MTT)法观察西罗莫司对白血病细胞HL60的体外生长抑制作用,蛋白质印迹法(Western-blot)检测西罗莫司处理HL60细胞后自噬相关蛋白LC3-Ⅱ的表达情况,流式细胞术(FCM)检测西罗莫司干预后HL60细胞的周期分布,并通过DNA琼脂糖凝胶电泳检测凋亡。结果四甲基偶氮唑蓝法结果显示,1、5、10、20、40nmol·L~(-1)的西罗莫司能够抑制HL60细胞增殖,并呈浓度依赖性(r=0.97,P<0.05)。蛋白质印迹法结果显示,各浓度组LC3-Ⅱ表达水平高于对照组(P<0.01),具有促进细胞自噬作用;FCM显示西罗莫司干预后的白血病细胞株出现细胞周期阻滞,G_0/G_1期细胞比例逐渐增加,S期细胞明显减少,与对照组比较具有显著性差异(P<0.05);琼脂糖凝胶电泳未见凋亡细胞形成DNA片段。结论西罗莫司具有抑制人急性早幼粒白血病细胞HL60增殖的作用,在限定浓度范围内以诱导HL60细胞发生自噬为主而不是凋亡。
Objective To investigate the effects of sirolimus on the proliferation, autophagy and apoptosis of human acute promyelocytic leukemia cell line HL60. Methods MTT assay was used to observe the inhibitory effect of sirolimus on HL60 cells in vitro. Western-blotting was used to detect the expression of autophagy-related protein LC3- Ⅱ, the distribution of HL60 cells after sirolimus intervention was detected by flow cytometry (FCM), and apoptosis was detected by DNA agarose gel electrophoresis. Results The results of MTT assay showed that sirolimus at 1, 5, 10, 20 and 40 nmol·L -1 could inhibit HL60 cell proliferation in a concentration-dependent manner (r = 0.97, P <0.05). The result of Western blot showed that the expression of LC3-Ⅱ in each concentration group was higher than that in control group (P <0.01), and promoted the autophagy. FCM showed cell cycle arrest in leukemia cell line after sirolimus intervention, / G_1 phase cells gradually increased, S phase cells decreased significantly, compared with the control group was significantly different (P <0.05); no apoptotic cells formed DNA fragments by agarose gel electrophoresis. Conclusion sirolimus can inhibit the proliferation of human acute promyelocytic leukemia HL60 cells in a concentration range to induce autophagy in HL60 cells rather than apoptosis.