华蟾素通过线粒体途径诱导鼻咽癌CNE-2细胞凋亡的机制  

Mechanism of Cinobufosin Inducing Apoptosis of Nasopharyngeal Carcinoma CNE-2 Cells Through Mitochondrial Pathway

作　　者：蔡庆春[1] 葛信艳 闫五玲 王白燕[2] 李瑞琴[1,3] 王莹莹 曾小虎 吕欢欢[1] CAI Qingchun;GE Xinyan;YAN Wuling;WANG Baiyan;LI Ruiqin;WANG Yingying;ZENG Xiaohu;Lü Huanhuan(Department of Pathology,the Third Affiliated Hospital of Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;School of Medicine,Henan University of Traditional Chiness Medicine,Zhengzhou 450046,China;Academy of Chiness Medicine,Henan University of Traditional Chiness Medicine,Zhengzhou 450046,China)

机构地区：[1]河南中医药大学第三附属医院病理科,河南郑州450046 [2]河南中医药大学医学院,河南郑州450046 [3]河南中医药大学中医药科学院,河南郑州450046

出　　处：《河南医学研究》2022年第8期1345-1350,共6页Henan Medical Research

基　　金：河南省科技攻关计划项目(2018ZY1003);河南省科技攻关计划项目(2019ZY2007)。

摘　　要：目的探讨华蟾素诱导人鼻咽癌CNE-2细胞增殖抑制、凋亡的作用机制。方法将人鼻咽癌CNE-2细胞分为阴性、阳性对照组及华蟾素给药组,采用四甲基偶氮唑蓝(MTT)法检测不同水平华蟾素对人鼻咽癌细胞的增殖抑制情况;吖啶橙/溴化乙锭(AO/EB)双荧光染色法观察药物对细胞形态的影响;流式细胞术(FCM)检测细胞凋亡率;免疫印迹法(Westernblot)检测线粒体凋亡途径相关蛋白细胞色素C(Cyt-C)和半胱氨酸天冬氨酸蛋白酶-9(caspase-9)的表达。结果MTT法检测显示,与阴性对照组相比,阳性对照组及华蟾素组可抑制鼻咽癌CNE-2细胞的增殖,倒置荧光显微镜下可见活细胞、凋亡细胞及坏死细胞,抑制率、凋亡/坏死细胞数量与药物剂量和作用时间呈正相关。FCM检测结果显示,华蟾素作用于CNE-2细胞24、48h后,各华蟾素组凋亡率均高于阴性对照组,且随着华蟾素药物水平升高、作用时间延长,凋亡率也升高,差异有统计学意义(P<0.05);Westernblot检测结果显示,与阴性对照组相比,阳性对照组和不同剂量的华蟾素组作用于CNE-2细胞24、48h后Cyt-C、caspase-9蛋白的表达增加,差异有统计学意义(P<0.05)。结论华蟾素对人鼻咽癌细胞CNE-2的增殖具有抑制作用,并能诱导鼻咽癌细胞凋亡,其机制可能是通过上调Cyt-C、caspase-9蛋白的表达实现的。Objective To explore the mechanism of cinobufosin inducing proliferation inhibition,apoptosis of human nasopharyngeal carcinoma CNE-2 cells.Methods Human nasopharyngeal carcinoma CNE-2 cells were grouped as follows:negative and positive control group,cinobufosin-administered group.The methyl thiazolyl tetrazolium(MTT)method was used to detect the proliferation of cinobufosin on CNE-2 cells.The growth and morphological changes of apoptotic cells was observed by acridine orange/ethidium bromide(AO/EB)dual fluorescent staining.Flow cytometry(FCM)technolog was used to detect cell apoptosis rate.Western blot technolog was used to detect the expression of mitochondrial apoptosis pathway related proteins cytochrome C(Cyt-C)and caspase-9 protein.Results The results of the MTT method showed that,compared with the negative control group,the positive control group and cinobufosin groups could inhibit the proliferation of CNE-2 cells.The living,apoptotic and necrotic cells were observed under the inverted light microscope.The inhibition rate of cell proliferation,the number of apoptotic/necrotic cells were positively correlated to medication dose and time.The results of FCM showed that the apoptosis rate of each cinoloufosin group were higher than that of the negative control group after cinobufosin acted on CNE-2 cells for 24 and 48 h.With increasing medication dose and time,the apoptotic rate also increased(P<0.05).Western blot test results showed that,compared with the negative control group,the expression of Cyt-C and caspase-9 protein increased in the positive control group and cinobufosin groups(P<0.05).Conclusion Cinobufosin can inhibit the proliferation of human nasopharyngeal carcinoma cells CNE-2 and induce apoptosis of nasopharyngeal carcinoma cells.The mechanism may be achieved by up-regulating the expression of Cyt-C and caspase-9 proteins.

关 键 词：华蟾素 鼻咽癌 线粒体途径 CNE-2细胞 凋亡 

分 类 号：R961[医药卫生—药理学]