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莲心碱对乳腺癌细胞MDA-MB-231自噬功能的影响

, PP. 629-633

Keywords: 乳腺癌,MDA-MB-,莲心碱,自噬

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Abstract:

目的观察莲心碱作用于乳腺癌细胞MDA-MB-231后,自噬表型的变化,初步论证莲心碱对乳腺癌细胞MDA-MB-231自噬功能的调控作用。方法透射电子显微镜观察莲心碱处理后MDA-MB-231的亚细胞结构变化,激光共聚焦显微镜观察pEGFP-LC3或ptfLC3(mRFP-EGFP-LC3)转染后自噬小体的形成和自噬流的变化,Westernblot检测自噬相关蛋白LC3B,SQSTM1/p62,Beclin1的表达,以及论证自噬流的变化。结果透射电镜显示,与对照组比较,莲心碱处理后MDA-MB-231细胞内有较多自噬空泡出现,部分分裂的线粒体,但未见到高密度的嗜锇性的自噬溶酶体结构;EGFP-LC3转染后,莲心碱组自噬体数量显著多于对照组(P<0.01);Westernblot检测到自噬相关蛋白LC3B-Ⅱ,SQSTM1/p62表达呈现量效性和时效性的增多[20μmol/L组或24h组与对照组比较(P<0.01)],Beclin1未见显著变化[20μmol/L组与对照组比较(P>0.05)];mRFP-EGFP-LC3转染后,对照组可见少量自噬体和自噬溶酶体,莲心碱和BafilomycinA1处理组均可见大量自噬体结构,未见显著自噬溶酶体结构;Westernblot证实莲心碱处理后增多的LC3B-Ⅱ不受BafilomycinA1预处理的影响[莲心碱组LC3B-Ⅱ,SQSTM1/p62蛋白水平与BafilomycinA1预处理加莲心碱组比较(P>0.05)]。结论莲心碱抑制自噬小体成熟,阻断乳腺癌细胞MDA-MB-231自噬降解作用。

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