天津医药

天津医药 ›› 2020, Vol. 48 ›› Issue (9): 801-806.doi: 10.11958/20200238

• 细胞与分子生物学 •    下一篇

不同浓度米诺环素对人胶质瘤细胞增殖、自噬、凋亡的影响及其机制

韩利民 1，严婉约 2，李巧巧 2，李科 2，刘丽 2，赵海龙 1△
  

  1. 1遵义医科大学病理生理学教研室（邮编563000）；2遵义医科大学第一临床学院
  • 收稿日期:2020-01-21 修回日期:2020-05-11 出版日期:2020-09-15 发布日期:2020-09-15
  • 通讯作者: 赵海龙 E-mail:Hailongzhao@zmu.edu.cn
  • 作者简介:韩利民（1988），男，硕士在读，主治医师，主要从事肿瘤药物靶点筛选方面研究
  • 基金资助:
    国家自然科学基金资助项目（31701007）；贵州省教育厅青年科技人才成长项目（2017-198）；贵州省大学生创新创业训练计划
    （    201710661042）

Effects of different concentrations of minocycline on the proliferation, autophagy and apoptosis of human glioma cells and its mechanism

HAN Li-min1, YAN Wan-yue2, LI Qiao-qiao2, LI Ke2, LIU Li2, ZHAO Hai-long1△ #br#   

  1. 1 Department of Pathophysiology, School of Basic Medical Sciences, Zunyi Medical University, Zunyi 563000, China;
    2 the First Clinical Institute of Zunyi Medical University
  • Received:2020-01-21 Revised:2020-05-11 Published:2020-09-15 Online:2020-09-15

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摘要：

摘要：目的 探讨不同浓度米诺环素对人类胶质瘤 U87 和 LN229 细胞增殖及凋亡的影响和作用机制。方法
（1）U87和LN229细胞设对照组（DMSO处理），5 μmol/L米诺环素组和10 μmol/L米诺环素组，分组处理72 h 后采用
MTT法检测细胞增殖水平，免疫荧光标记检测细胞自噬蛋白微管相关蛋白l轻链3B亚基（LC3B）表达水平，Western
blot法检测细胞自噬和凋亡相关蛋白的表达变化。（2）构建沉默信息调节因子2相关酶1（SIRT1）-shRNA载体，观察
敲低SIRT1表达后，不同浓度米诺环素对U87和LN229细胞自噬的影响。结果 （1）与对照组相比，5 μmol/L和10
μmol/L米诺环素组细胞增殖能力下降，免疫荧光标记显示胞浆内自噬标记蛋白LC3B的表达增多，Western blot结果
显示哺乳动物雷帕霉素靶蛋白（mTOR）水平显著降低，自噬基因相关蛋白5（Atg5）、磷酸化AMP依赖的蛋白激酶α亚
基（p-AMPKα）、SIRT1水平显著增加（P＜0.05）。与对照组相比，10 μmol/L米诺环素组B淋巴细胞瘤-2（Bcl-2）、磷
酸化 p70 核糖体蛋白 S6 激酶（p-p70s6k）水平显著降低，活化形式的 Caspase-3（Cleaved caspase-3）水平显著增加
（P＜0.05），而5 μmol/L米诺环素组除Cleaved Caspase-3表达水平下降外其他凋亡相关蛋白水平未见显著变化（P＞
0.05）。（2）敲低SIRT1表达后，shSIRT1组mTOR和LC3B表达水平较对照组明显下降；而经过米诺环素处理后，mTOR
的表达出现明显升高，而LC3B表达水平仅部分恢复。结论 5、10 μmol/L的米诺环素均能有效抑制人类胶质瘤U87
和LN229细胞的生长，其机制可能与AMPK/SIRT1通路诱导细胞自噬和p70s6k/Bcl-2通路诱导细胞凋亡有关。

关键词: 米诺环素, 神经胶质瘤, 细胞增殖, 细胞凋亡, 自噬, AMP活化蛋白激酶类, 沉默信息调节因子2相关酶1

Abstract:

Abstract：Objective To investigate the effects and mechanism of minocycline at different concentrations on the
proliferation and apoptosis of human glioma U87 and LN229 cells. Methods (1) U87 and LN229 cells were divided into
three groups: control group (DMSO treatment), 5 μmol/L minocycline group and 10 μmol/L minocycline group. After 72
hours of treatment, the level of cell proliferation was detected by MTT method, the expression of autophagy LC3B was
detected by immunofluorescence labeling and the expressions of autophagy and apoptosis-related proteins were detected by
Western blot assay. (2) SIRT1-shRNA vector was constructed to observe the effects of different concentrations of
minocycline on the autophagy of U87 and LN229 cells after knockdown of SIRT1 expression. Results (1) Compared with
the control group, the MTT results showed that the proliferation of glioma cells was significantly inhibited in 5 μmol/L and 10
μmol/L minocycline groups, and immunofluorescence staining showed that the development of autophagy labeled protein
LC3B was increased. Western blot results showed that the level of mTOR was significantly decreased, and the levels of Atg5,
p-AMPK α and SIRT1 were significantly increased (P＜0.05). Western blot assay showed that the levels of Bcl-2 and pp70s6k were significantly decreased and the activated Caspase-3 level was significantly increased in 10 μmol/L minocycline group compared with those of control group (P＜0.05), but there were no significant changes in the levels of Bcl-2 and pp70s6k proteins in 5 μmol/L minocycline group (P＞0.05). (2) After knocking down the expression of SIRT1, the expression
levels of mTOR and LC3B were significantly lower in shSIRT1 group than those of control group, while after minocycline
treatment, the expression of mTOR increased significantly, while the expression level of LC3B only partially recovered.
Conclusion Minocycline of 5 and 10 μmol/L can effectively inhibit the growth of human glioma U87 and LN229 cells,
which may be related to the AMPK/SIRT1 pathway induced cell autophagy and p70s6k/Bcl-2 pathway induced apoptosis.

Key words: minocycline, glioma, cell proliferation, apoptosis, autophagy, AMP-activated protein kinases, SIRT1

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