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[目的]分析自噬在熊果酸抑制人宫颈癌He La细胞增殖中的作用。[方法]体外培养人宫颈癌He La细胞,不同浓度(5、10、20μmol/L)的熊果酸处理48h后,采用MTT法检测He La细胞增殖抑制率的变化;透射电镜观察细胞自噬超微结构的改变;Western blotting检测自噬相关蛋白p62及Beclin-1的表达情况;微管相关蛋白1轻链3A/B(microtubule-associated protein 1 light chain 3 A/B,LC3A/B)免疫荧光检测熊果酸对He La细胞自噬水平的影响;检测自噬抑制剂3-甲基腺嘌呤(3-methyladenine,3-MA)抑制自噬前后对熊果酸增殖抑制作用的影响。[结果]不同浓度(5、10、20μmol/L)的熊果酸处理组对He La细胞的抑制率分别为20.1%±1.3%、35.6%±2.6%和49.0%±1.0%,熊果酸可抑制He La细胞增殖并呈剂量依赖性(F=446.177,P<0.001);熊果酸能诱导He La细胞发生自噬:透射电镜观察发现经熊果酸处理后He La细胞中自噬囊泡明显增加;Western blotting分析表明熊果酸可呈剂量依赖性增加Beclin-1的表达,降低p62的表达;免疫荧光检测显示熊果酸处理后,He La细胞的荧光强度与对照组相比明显增强;3-MA与熊果酸联合作用于He La细胞时可抑制细胞增殖。[结论]熊果酸抑制He La细胞增殖,并诱导其发生自噬,自噬抑制剂3-MA能够增强熊果酸对He La细胞的增殖抑制作用。
[Objective] To analyze the role of autophagy in inhibiting the proliferation of human cervical cancer He La cells by ursolic acid. [Methods] Human cervical cancer He La cells were cultured in vitro. After treated with ursolic acid (5, 10, 20μmol / L) for 48h, MTT assay was used to detect the proliferation inhibition rate of He La cells. The autophagy The changes of ultrastructure were observed. The expression of autophagy-related proteins p62 and Beclin-1 was detected by Western blotting. The microtubule-associated protein 1 light chain 3A / B (LC3A / B) The effect of ursolic acid on the autophagy of HeLa cells was detected by fluorescence assay. The effect of 3-methyladenine (3-MA), an inhibitor of autophagy, on the inhibition of ursolic acid proliferation was examined. [Result] The inhibitory rates of UA at different concentrations (5, 10 and 20μmol / L) on HeLa cells were 20.1% ± 1.3%, 35.6% ± 2.6% and 49.0% ± 1.0% Inhibited the proliferation of HeLa cells in a dose-dependent manner (F = 446.177, P <0.001). UA induced the autophagy in HeLa cells. The results of transmission electron microscopy showed that ursolic acid The results of Western blotting showed that ursolic acid increased the expression of Beclin-1 in a dose-dependent manner and decreased the expression of p62. The immunofluorescence showed that the fluorescence intensity of HeLa cells was significantly higher than that of the control group Enhanced; 3-MA and ursolic acid combined inhibit the proliferation of He La cells. [Conclusion] UA inhibited Hela cell proliferation and induced its autophagy. Autophagy inhibitor 3-MA could enhance the inhibitory effect of ursolic acid on He La cell proliferation.