摘要
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MDAMB-231的亚细胞结构变化,激光共聚焦显微镜观察p EGFP-LC3或ptf LC3(mRFP-EGFP-LC3)转染后自噬小体的形成和自噬流的变化,Western blot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);Western blot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24 h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Bafilomycin A1处理组均可见大量自噬体结构,未见显著自噬溶酶体结构;Western blot证实莲心碱处理后增多的LC3B-Ⅱ不受Bafilomycin A1预处理的影响[莲心碱组LC3B-Ⅱ,SQSTM1/p62蛋白水平与Bafilomycin A1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。
Objective To determine the effect of liensinine on the autophagic phenotypes in breast cancer MDA-MB-231 cells in order to investigate its regulative role on autophagic functions in the cells. Methods Transmission electron microscopy （TEM） was used to observe the uhrastructural changes in MDA-MB-231 cells after liensinine treatment （20 μmol/L for 24 h）. Confocal laser scanning microscopy was employed to determine the formation of autophagosome and autophagic flux after the cells were transfected with pEGFP-LC3 or pffLC3. Western blot analysis was used to detect the expression of autophagy related proteins, including LC3B, SQSTM1/p62 and Beclinl. Results TEM displayed there were more autophagic vacuoles and some fission mitochondria, but no high density osmiophilic autolysosomes in the MDA-MB-231 cells after liensinine treatment. Confocal laser scanning microscopy confirmed that there were more EGFP-LC3 puncta in the cells after pEGFP-LC3 transfection （ P 〈 0.01 ）. Western blotting indicated that the expression levels of LC3B-Ⅱ and SQSTM1/p62 were increased in a dose- and time-dependent manner （20 μmol/L or 24 h vs control, P 〈0.01 ） , and those of LC3B- I and Beclinl had no obvious change （20 Ixmol/L vs control, P 〉 0. 05 ） in the cells after pEGFP-LC3 transfection. While, in the cells after mRFP-EGFP-LC3, there were only a few of autophagosomes and autolysosomes in the control cells, but a lot of autophagosomes in the cells treated with liensinine and bafilomycin A1, but no autolysosomes. no effect on liensinine induced enhancement of LC3B-II Western blotting indicated that bafilomycin A1 had and SQSTM1/p62 （P 〉 0.05）. Conclusion Liensinine suppresses the maturation of autophagosome, and therefore blocks the autophagic degradation in MDA-MB-231 cells.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2015年第7期629-633,共5页
Journal of Third Military Medical University
基金
国家自然科学基金青年科学基金(81402202)~~
