左旋一叶萩碱诱导人非小细胞肺癌A549细胞自噬机制的研究

L-Securinine induced the human non small cell lung cancer A549 cell autophagy and its molecular mechanism

目的:研究左旋一叶萩碱诱导人非小细胞肺癌A549细胞自噬的机制。方法:常规体外培养人非小细胞肺癌A549细胞株,取对数生长期的细胞传代24h后加入不同浓度(6.25、12.5、25、50、100、200μmol/L)左旋一叶萩碱处理,分别继续培养24、36、48h后,用CCK-8法测定细胞增殖活性;倒置显微镜下观察对照组和各实验组的细胞形态变化;分别用不同浓度(0、20μmol/L)左旋一叶萩碱作用A549细胞48h后,进行单丹磺酰尸胺(monodansylcadaverine,MDC)染色,分别通过荧光显微镜检测细胞自噬发生情况;荧光定量RT-PCR法检测Beclin 1在mRNA水平表达变化情况。结果:左旋一叶萩碱可以抑制A549细胞增殖,并且呈浓度和时间依赖性(P<0.05)。其中,21.204μmol/L左旋一叶萩碱作用A549细胞48h后,细胞死亡率接近50%;倒置显微镜观察到药物作用后细胞形态发生了明显变化;随着左旋一叶萩碱剂量增加,MDC阳性细胞的荧光强度及细胞内MDC荧光颗粒数目明显增加。另外,研究还发现随着药物浓度的上升,自噬基因Beclin 1的表达明显增强。结论:左旋一叶萩碱具有抑制人非小细胞肺癌A549细胞增殖、诱导细胞自噬性凋亡,其作用具有浓度和时间依赖性。左旋一叶萩碱上调自噬基因Beclin 1 mRNA的表达水平可能是其诱导A549细胞自噬性凋亡的机制之一。

AIM： To explore the mechanism of L-securinine induced autophagy of human non small cell lung cancer A549 cell autophagy. METHODS： A549 cell lines were cultured in vitro and L-securinine at different concentrations （6.25,12.5,25,50,100,200 μmol/L） was added 24 h after cell lines were transferred. Then the plates were cultured for 24, 36 and 48 h. CCK-8 method was used to detect the antitumor effect of human non small cell lung cancer A549 in vitro. Inverted microscope was used to observe A549 cells treated with L-securinine morphologi- cal changes. The A549 cells were treated with different concentrations （0, 20 μmol/L） of L-se- curinine for 24 h. Then autophagy was analyzed by fluorescence microscope following staining with monodansylcadaverine （MDC）. The mR- NA levels of Beclin-1 were detected using real- time RT-PCR pre and post-treatment of L-se- curinine. RESULTS： The generation depressioneffects of A549 cells cultured in vitro were de- tected by CCK-8 method （P〈0.05）, and there were dosage-time dependent relationships. The morphology of cells become small and round, the process of cell division got less were ob- served. L-securinine-treated cells exhibited higher fluorescent density and more MDC-la- beled particles in A549 cells compared with the