摘要
目的:以人肝癌HepG_(2)细胞为研究对象,探讨青藤碱诱导人肝癌HepG_(2)细胞凋亡的作用机制。方法:采用CCK-8法检测青藤碱对人肝癌HepG_(2)细胞的增殖抑制作用;采用倒置显微镜、荧光显微镜观察人肝癌HepG_(2)细胞形态变化;采用流式细胞仪检测人肝癌HepG_(2)细胞的凋亡率;采用试剂盒检测肿瘤细胞膜总蛋白、胆固醇、唾液酸含量;采用荧光染色法检测肿瘤细胞膜微黏度、流动性、封闭度;采用ATP酶试剂盒检测肿瘤细胞膜Na^(+)-K^(+)ATP酶和Ca^(2+)-Mg^(2+)ATP酶含量;采用流式细胞仪检测线粒体MPTP转换孔活性、线粒体跨膜电位变化;采用试剂盒检测Caspase-3蛋白水平。结果:通过CCK-8法检测发现青藤碱对人肝癌HepG_(2)细胞具有增殖抑制作用,作用72 h的IC_(50)为1.4 mmol/L。倒置显微镜、荧光显微镜观察细胞形态发现青藤碱各组细胞出现凋亡迹象。采用试剂盒检测肿瘤细胞膜主要组成成分、结构变化情况发现,青藤碱能降低人肝癌HepG_(2)细胞膜总蛋白、胆固醇、唾液酸含量,并且能使肿瘤细胞膜微黏度显著升高,流动性、封闭度显著降低;检测青藤碱对人肝癌HepG_(2)细胞膜ATP酶含量影响发现,青藤碱能显著降低人肝癌HepG_(2)细胞Na^(+)-K^(+)ATP酶和Ca^(2+)-Mg^(2+)ATP酶含量,且其作用呈浓度依赖性;检测青藤碱对人肝癌HepG_(2)细胞线粒体膜MPTP转换孔、线粒体膜电位的影响发现青藤碱能使人肝癌HepG_(2)细胞线粒体膜MPTP转换孔通透性增加,同时能升高肿瘤细胞内Caspase-3蛋白表达,且其作用呈浓度依赖性。结论:青藤碱对人肝癌HepG_(2)细胞具有增殖抑制作用,并能通过干扰肿瘤细胞膜受体,破坏肿瘤细胞膜组成结构功能,激活肿瘤细胞凋亡的相关因子,进而启动细胞凋亡程序。
Objective:To investigate the mechanism of sinomenine in inducing apoptosis of human hepatocellular carcinoma HepG_(2)cells by using human hepatocellular carcinoma HepG_(2)cells as research objects.Methods:The inhibitory effect of sinomenine on proliferation of human hepatocellular carcinoma HepG_(2)cells was detected by CCK-8 method.Morphological changes of human hepatocellular carcinoma HepG_(2)cells were observed by inverted microscope and fluorescence microscope.Flow cytometry was used to detect the apoptosis rate of human hepatocellular carcinoma HepG_(2)cells.The contents of total protein,cholesterol and sialic acid in tumor cell membrane were detected by the kit.Fluorescence staining was used to detect the microviscosity,fluidity and sealing degree of tumor cell membrane.The concents of Na^(+)-K^(+)ATP enzyme and Ca^(2+)-Mg^(2+)ATP enzyme in tumor cell membrane were detected by ATP enzyme kit.Flow cytometry was used to detect the activities of mitochondrial MPTP transfer hole and the changes of mitochondrial transmembrane potential.The Caspase-3 protein level was detected by the kit.Results:It was found that sinomenine inhibited the proliferation of human hepatocellular carcinoma HepG_(2)cells by CCK-8 assay with IC_(50)of 1.4 mmol/L after 72 hours treatment.The morphology of the cells was observed under inverted microscope and fluorescence microscope,and the apoptosis of the cells in each sinomenine group was found.Using the kit to detect the changes in the main components and structures of tumor cell membranes,sinomenine could reduce the contents of total protein,cholesterol and sialic acid of cell membrane of human hepatocellular carcinoma HepG_(2)cells,and significantly increase the microviscosity and reduce the fluidity and sealing degree of tumor cell membrane.It was found that sinomenine significantly reduced the Na^(+)-K^(+)ATP enzyme and Ca^(2+)-Mg^(2+)ATP enzyme concents in human hepatocellular carcinoma HepG_(2)cells,and the effect was concentration dependent.The effects of sinomenine on MPTP transformation hole and mitochondrial membrane potential of human hepatocellular carcinoma HepG_(2)cells were detected.It was found that sinomenine could increase the permeability of MPTP transformation hole in the mitochondrial membrane of human hepatocellular carcinoma HepG_(2)cells,and increase the expression of Caspase-3 protein in tumor cells,its effect was concentration dependent.Conclusion:Sinomenine can inhibit the proliferation of human hepatocellular carcinoma HepG_(2)cells,and start the apoptosis process by interfering with tumor cell membrane receptors,destroying the structure and function of tumor cell membrane and activating the related factors of tumor cell apoptosis.
作者
李吉业
辛国松
于淼
高世勇
于蕾
郎朗
季宇彬
LI Ji-ye;XIN Guo-song;YU Miao;GAO Shi-yong;YU Lei;LANG Lang;JI Yu-bin(Engineering Research Center for Medicine,Harbin University of Commerce,Harbin 150076,China;National Ministry of Education Anti-tumor Natural Drug Engineering Research Center,Harbin 150076,China)
出处
《中药材》
CAS
北大核心
2021年第3期667-673,共7页
Journal of Chinese Medicinal Materials
基金
中国博士后面上项目(2019M651296)
黑龙江省自然科学基金联合引导项目(LH2019H066)
哈尔滨商业大学青年创新人才项目(2019CX37)
关键词
青藤碱
抗肿瘤
肝癌
细胞膜
细胞凋亡
Sinomenine
Anti-tumor
Hepatocellular carcinoma
Cell membrane
Apoptosis
作者简介
李吉业(1994-),男,在读硕士研究生,专业方向:抗肿瘤天然药物研究,Tel:13101660880,E-mail:8537044@qq.com;通讯作者:辛国松,E-mail:13766801150@163.com。
