细胞自噬对高三尖杉酯碱诱导K562细胞凋亡效应的影响

发布时间：2018-05-07 01:36

  本文选题：高三尖杉酯碱 + K562细胞　； 参考：《南京医科大学》2014年硕士论文

【摘要】：目的观察经高三尖杉酯碱(Homoharringtonine, HHT)处理后的不同时间点K562细胞凋亡率的变化。方法分别采用MTT、流式细胞术和western blot等方法检测HHT处理后的不同时间点K562细胞的增殖活力、凋亡率及Bcl-2蛋白和Caspase-3蛋白的表达。结果①HHT (终浓度为lOng/ml)作用后, K562细胞增殖活力逐渐下降,但作用后期细胞增殖活力又逐渐升高,第1天到第8天的细胞增殖活力分别为(93.6±1.2)%、(81.3±0.9)%、(69.8+0.7)%、(57.2±2.1)%、(43.8+1.1)%、(54.1+1.4)%、(64.5±1.3)%和(70.6+1.8)%。与之相对应的是K562细胞凋亡率在HHT作用早期逐步上升而在作用后期逐渐下降。②HHT处理后,K562细胞的激活型caspase-3蛋白(17KD及19KD)表达从第1天至第7天逐渐增高,第8天开始下降(p0.05)。③HHT处理后1-8天K562细胞Bcl-2阳性表达呈现先低后高的特点：前7天逐渐下降,第8天开始上升,差异有统计学意义(p0.05)结论在体外处理K562细胞的过程中,HHT的细胞凋亡诱导作用在体外培养早期较强,但在作用后期凋亡诱导作用明显减弱。目的 观察单用高三尖杉酯碱(Homoharring-tonine,HHT)及HHT联合自噬抑制剂3-MA对K562细胞增殖、凋亡以及自噬的影响。方法10ng/ml的HHT及1.5mmol/L的3-MA作用K562细胞后,检测细胞Beclinl基因表达(RT-PCR法)、LC3 Ⅱ/Ⅰ及caspase-3凋亡蛋白表达(Western blot印迹法)和细胞自噬(透射电子显微镜)。结果 ①HHT处理后,K562细胞的Beclin 1基因和LC3 Ⅱ/Ⅰ表达水平在作用的早期(分别为d1-d5和d1-d6)逐步下降,但HHT作用后期(分别为d6-d8和d7-d8)两者的表达水平开始上升；②caspase-3蛋白表达水平在HHT作用早期(d1-d7)逐步升高,d8开始下降。③HHT联用3-MA组的Beclin 1基因及LC3II/I表达水平在d1-d8逐渐下降,无后期升高现象发生。④HHT联用3-MA组的caspas e-3蛋白表达水平在d1--d8内持续逐渐升高。⑤电镜下,HHT作用的第8天K562细胞自噬体显著增多,而HHT联合3-MA作用第8天无自噬体出现。结论HHT可诱导K562细胞凋亡,但HHT持续后,K562细胞可发生自噬而使HHT的细胞凋亡诱导作用减弱；联用自噬抑制剂可增强HHT的细胞凋亡诱导作用。
[Abstract]:Objective to observe the apoptosis rate of K562 cells treated with homoharringtonine (HHT) at different time points. Methods MTT, flow cytometry and western blot were used to detect the proliferation activity, apoptosis rate and the expression of Bcl-2 protein and Caspase-3 protein in K562 cells treated with HHT at different time points. Results the proliferative activity of K562 cells decreased gradually after treatment with 1HHT (final concentration was lOng / ml), but the proliferative activity of K562 cells increased gradually in the late stage of treatment. The proliferative activities of K562 cells were 93.6 卤1.29.80.73 卤0.99.70.78 from day 1 to day 8, respectively. The proliferative activity of K562 cells was 54.2 卤2.11g / ml and 64.5 卤1.3g% and 70.6 / 1.8U / d, respectively. In contrast, the apoptotic rate of K562 cells increased gradually at the early stage of HHT treatment, but decreased gradually at the late stage of treatment. The expression of activated caspase-3 protein (17KD and 19KD) in K562 cells increased gradually from day 1 to day 7 after treatment with .2HHT. On the 8th day, the expression of Bcl-2 in K562 cells decreased at first and then increased after treatment with HHT. The expression of Bcl-2 decreased gradually at the first 7 days, and then increased on the 8th day. Conclusion the apoptosis induction of HHT in K562 cells was stronger at the early stage of culture in vitro, but the apoptotic effect was significantly weakened in the late stage of treatment of K562 cells. Objective to observe the effects of homoharringtonine (HHT) and HHT combined with autophagy inhibitor 3-MA on the proliferation, apoptosis and autophagy of K562 cells. Methods after K562 cells were treated with HHT of 10ng/ml and 3-MA of 1.5mmol/L, the expression of Beclinl gene was detected by RT-PCR and the expression of caspase-3 apoptosis protein was detected by Western blot and autophagy (transmission electron microscope). Results after 1HHT treatment, the expression of Beclin 1 gene and LC3 鈪，

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