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蓝萼甲素通过Bcl-2/Beclin1靶点调节人肝癌HCCLM3细胞自噬

与凋亡的机制研究 Δ

朱琳琳 ，张明明，郭格格，徐祉轩（新乡医学院医学检验学院/河南省分子检验与医学检验技术协同创新中心/河
＊
南省免疫与靶向药物重点实验室，河南 新乡 453003）

中图分类号 R965；R735.7 文献标志码 A 文章编号 1001-0408（2022）09-1082-06
DOI 10.6039/j.issn.1001-0408.2022.09.10

摘 要 目的 研究蓝萼甲素（GLA）对人肝癌HCCLM3细胞自噬与凋亡的调节作用机制。方法 HCCLM3细胞按不同实验目的主
要设对照组、GLA 2.5 μg/mL组、GLA 5 μg/mL组、GLA 10 μg/mL组。对照组仅加入完全培养基，各给药组分别加入含GLA相应终
质量浓度的完全培养基。采用流式细胞术检测细胞周期分布与凋亡情况；采用透射电镜观察细胞线粒体形态和自噬情况（仅设对
照组、GLA 5 μg/mL组）；采用JC-1染色和荧光倒置显微镜观察并检测细胞线粒体膜电位；采用Western blot法检测细胞中Bcl-2、
Bax、Beclin1、活性半胱氨酸天冬氨酸蛋白酶3（cleaved caspase-3）蛋白的表达；采用免疫共沉淀法检测细胞中Bcl-2与Beclin1的结
合与解离（仅设GLA 5 μg/mL组、GLA 10 μg/mL组）。结果 与对照组相比，GLA 5、10 μg/mL能够诱发细胞周期显著阻滞于G2～M
期、诱导线粒体膜电位显著下降、增加促凋亡作用，还能显著促进Bax、cleaved caspase-3、Beclin1蛋白表达，显著抑制Bcl-2蛋白表
达（P＜0.01）；GLA 5 μg/mL还可引发线粒体形态显著改变，自噬小体增多。免疫共沉淀法结果显示，与GLA 5 μg/mL比较，GLA
10 μg/mL能够增强Bcl-2与Beclin1的结合。结论 GLA可能通过Bcl-2/Beclin1靶点调节HCCLM3细胞自噬与凋亡，且作用效果与
剂量密切相关。
关键词 蓝萼甲素；靶点；机制；自噬；凋亡；Bcl-2；Beclin1

Study on regulation mechanism of glaucocalyxin A on the autophagy and apoptosis of HCCLM3
hepatocellular carcinoma cells through Bcl-2/Beclin1 target
ZHU Linlin，ZHANG Mingming，GUO Gege，XU Zhixuan（School of Laboratory Medicine，Xinxiang Medical
University/Henan Collaborative Innovation Center of Molecular Diagnosis and Laboratory Medicine/Henan Key
Laboratory of Immunology and Targeted Therapy，Henan Xinxiang 453003，China）

ABSTRACT OBJECTIVE To study the regulatory mechanism of glaucocalyxin A （GLA） on autophagy and apoptosis of
HCCLM3 hepatocellular carcinoma cells. METHODS HCCLM3 cells were taken，and control group，GLA 2.5 μg/mL group，GLA
5 μg/mL group and GLA 10 μg/mL group were mainly set according to different experimental purposes. In control group，only
complete medium was added；in each administration group，complete medium containing the corresponding final concentration of
GLA was added. Cell cycle distribution and apoptosis were detected by flow cytometry；mitochondrial morphology and autophagy
were observed by transmission electron microscope（only control group，GLA 5 μg/mL group）；JC-1 staining and fluorescence
inverted microscope were used to observe and detect the mitochondrial membrane potential of the cells；Western blot assay was
used to detect the protein expression of Bcl-2， Bax， Beclin1 and cleaved caspase-3 proteins in the cells； the
co-immunoprecipitation method was used to detect the binding and dissociation of Bcl-2 and Beclin1（only GLA 5 μg/mL group，
GLA 10 μg/mL group）. RESULTS Compared with control group，GLA 5 μg/mL and GLA 10 μg/mL could induce a significant
arrest of the cell cycle in the G2-M phase for HCCLM3，a significant decrease in mitochondrial membrane potential，an increase in
apoptosis as well as significant promotion of the protein expression of Bax，cleaved caspase-3 and Beclin1，and significant
inhibition of the protein expression of Bcl-2（P＜0.01）. GLA 5 μg/mL also significantly changed mitochondrial morphology and
increased autophagosomes. The results of co-immunoprecipitation showed that compared with GLA 5 μg/mL，GLA 10 μg/mL could
enhance the binding of Bcl-2 and Beclin1. CONCLUSIONS GLA can regulate the autophagy and apoptosis of HCCLM3 cells by
Bcl-2/Beclin1 target. The effect is closely related to the dose of GLA.
KEYWORDS glaucocalyxin A；target；mechanism；autophagy；apoptosis；Bcl-2；Beclin1

细胞自噬与凋亡存在诸多联系，与一系列基因激
Δ 基金项目：河南省科技攻关项目（No.212102310753）
活、表达调控相关。正常情况下，两者处于平衡状态；药
＊副教授，博士。研究方向：中草药抗肿瘤作用机制。电话：
0373-3832181。E-mail：linbetter@163.com 物、环境可以打破二者平衡，引发细胞走向不同结局，从

·1082 · China Pharmacy 2022 Vol. 33 No. 9 中国药房 2022年第33卷第9期