姜黄素诱导人髓母细胞瘤Daoy细胞自噬及其机制研究

发布时间：2018-08-02 19:28

【摘要】：目的：观察姜黄素（Curcumin）诱导人髓母细胞瘤Daoy细胞自噬的作用，并探讨其可能机制。 方法：体外培养人髓母细胞瘤Daoy细胞，透射电镜检测姜黄素处理前后Daoy细胞超微结构变化，免疫荧光染色、RT-PCR和Westernblot检测自噬相关蛋白Beclin1和LC3的表达；Western blot检测PI3K/Akt/mTOR信号通路中PI3K、p-Akt和p-mTOR的表达；姜黄素分别与自噬抑制剂wortmannin、PI3K激动剂insulin联合处理细胞后，Western blot检测自噬相关蛋白Beclin1和LC3及PI3K/Akt/mTOR信号通路相关蛋白的表达变化。 结果：透射电镜结果显示，随姜黄素作用时间增加，自噬体明显增多。免疫荧光染色显示Beclin1和LC3在姜黄素处理组中的表达强度明显高于对照组。RT-PCR显示与对照组相比，Beclin1mRNA和LC3mRNA的表达在处理组(24h和48h)明显增强，差异有统计学意义（F=1360.962，42.366，P 0.05）。Western blot显示处理组（24h和48h）Beclin1、LC3-Ⅱ/LC3-Ⅰ的表达明显高于对照组（0h）,而PI3K、p-Akt和p-mTOR的表达明显低于对照组，差异有统计学意义（F=32.723,11.847，，329.662,80.638，183.536，P 0.05）。自噬抑制剂wortmannin与姜黄素联合处理后不能降低姜黄素诱导的自噬。PI3K激动剂insulin与姜黄素联合处理后不能有效逆转姜黄素对PI3K/Akt/mTOR信号通路的抑制作用。 结论：姜黄素通过诱导自噬抑制髓母细胞瘤Daoy细胞生长,其机制可能与上调自噬相关基因表达，抑制自噬负调控PI3k/Akt/mTOR信号通路有关。
[Abstract]:Aim: to investigate the effect of curcumin (Curcumin) on autophagy of human myeloblastoma Daoy cells and its possible mechanism. Methods: human myeloblastoma Daoy cells were cultured in vitro. Ultrastructural changes of Daoy cells before and after curcumin treatment were detected by transmission electron microscope. The expression of autophagy associated protein Beclin1 and LC3 was detected by RT-PCR and Westernblot. Western blot was used to detect the expression of PI3KP- Akt and p-mTOR in PI3K/Akt/mTOR signaling pathway, and curcumin combined with wortmanninnine PI3K agonist insulin was used to detect the expression of Beclin1, LC3 and PI3K/Akt/mTOR signal pathway related proteins. Results: the results of transmission electron microscope showed that the autophagy increased with the increase of curcumin action time. Immunofluorescence staining showed that the expression of Beclin1 and LC3 in curcumin treated group was significantly higher than that in control group. RT-PCR showed that the expression of Beclin1 mRNA and LC3mRNA in the treatment group (24h and 48h) was significantly higher than that in the control group. The expression of Beclin1 + LC3- 鈪

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