莲心碱对乳腺癌细胞MDA-MB-231自噬功能的影响-第三军医大学学报.docVIP

莲心碱对乳腺癌细胞MDA-MB-231自噬功能的影响 周 静1，李国兵1，郑 怡1，银 涛2，高 宁1 （400038重庆，第三军医大学药学院生药学教研室1；510507广州，广州军区疾病预防控制中心2） ［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，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LC3荧光团数量显著多于对照组（p0.001）；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（20μM组或24小时组与对照组比较p0.001），LC3B-Ⅰ，Beclin1未见显著变化（20uM组与对照组比较p0.05）；mRFP-EGFP-LC3转染后，对照组呈现胞质弥散的黄色荧光，处理组呈现黄色LC3荧光团结构，未见显著红色荧光团结构；Western blot论证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p0.05)。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。 ［关键词］ 乳腺癌；MDA-MB-231；莲心碱；自噬 ［中图法分类号］ R285.5；R737.9 [文献标志码] A The effect of liensinine on autophagic process in breast cancer cells MDA-MB-231 Zhou Jing1, Li Guobing1， Zheng Yi1, Yin Tao2, Gao Ning1 (1Third Military Medical University, Chongqing, 400038, China; 2Center for Disease Control and Prevention of Guangzhou Military Area, Guangzhou, 510507, China) ［Abstract］ Objective Observe the autophagic changing in breast cancer MDA-MB-231 cells after treating with liensinine, and determine the regulation of liensinine on autophagic function in MDA-MB-231 cells. Methods Using transmission electron microscope (TEM) to observe the ultrastructural changes in MDA-MB-231 cells treated with liensinine. After transfecting with pEGFP-LC3 or ptfLC3 in MDA-MB-231 cells, a confocal laser scanning microscope was used to determine the formation of autophagosome and autophagic flux. Western blot was used to detect the expressing of autophagy related proteins. Results TEM showed significant structure of autophagic vacuole accumulating in MDA-MB-231 cells treated with liensinine, but lacking high density structure with single membrane which is standard feature of autolysosome. Confocal mi