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
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篇名: 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
TITLE: Study on regulation mechanism of glaucocalyxin A on the autophagy and apoptosis of HCCLM 3 hepatocellular carcinoma cells through Bcl- 2/Beclin1 target
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、GLA2.5μg/mL组、GLA5μg/mL组、GLA10μg/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、GLA5μg/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Westernblot法检测细胞中Bcl-2、Bax、Beclin1、活性半胱氨酸天冬氨酸蛋白酶3(cleavedcaspase-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GLA5μg/mL组、GLA10μg/mL组)。结果与对照组相比,GLA5、10μg/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、cleavedcaspase-3、Beclin1蛋白表达,显著抑制Bcl-2蛋白表达(P<0.01);GLA5μg/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GLA5μg/mL比较,GLA10μg/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。
ABSTRACT: OBJECTIVE To study the regulator y mech anism 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 Beclin 1(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 G 2-M phase for HCCLM 3,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 Beclin 1,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 Beclin 1. CONCLUSIONS GLA can regulate the autophagy and apoptosis of HCCLM 3 cells by Bcl-2/Beclin1 target. The effect is closely related to the dose of GLA.
期刊: 2022年第33卷第09期
作者: 朱琳琳,张明明,郭格格,徐祉轩
AUTHORS: ZHU Linlin,ZHANG Mingming,GUO Gege,XU Zhixuan
关键字: 蓝萼甲素;靶点;机制;自噬;凋亡;Bcl-2;Beclin1
KEYWORDS: glaucocalyxin A ;target;mechanism;autophagy;apoptosis;Bcl-2;Beclin1
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