去甲斑蝥素诱导黑色素瘤M14细胞凋亡及相关机制的研究  

Norcantharidin induced apoptosis of melanoma M14 cells and its action mechanism

作　　者：高杨[1] 丛力宁 程毅[2] 刘岩[1] GAO Yang;CONG Lining;CHENG Yi(Department of Interventional Radiology, Hebei Provincial Children’s Hospital,Hebei,Shijiazhuang 050000,China)

机构地区：[1]河北医科大学附属河北省儿童医院介入科,石家庄市050011 [2]河北医科大学第四医院皮肤科

出　　处：《河北医药》2020年第2期179-183,共5页Hebei Medical Journal

基　　金：河北省中医药管理局科研计划(编号:2017215)

摘　　要：目的探讨去甲斑蝥素对人黑色素瘤M14细胞增殖和凋亡的影响及其作用机制。方法采用CCK8法检测不同浓度去甲斑蝥素对黑素瘤M14细胞增殖的影响。实时荧光半定量PCR(RT-PCR)方法检测半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)、Capase-9、Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤/白血病-2(Bcl-2)、细胞色素c基因的mRNA的表达,以2’,7’-二乙酰二氯荧光素(DCFH-DA)为荧光探针检测不同浓度去甲斑蝥素对细胞内活性氧(ROS)产生的影响,双重加热测定法检测对细胞内丙二醛(MDA)水平的影响。结果CCK8实验显示去甲斑蝥素抑制黑色素瘤M14细胞增殖并诱导其凋亡,呈浓度依赖性。RT-PCR结果显示去甲斑蝥素可诱导M14细胞内凋亡相关基因Caspase-3、Capase-9、Bax、细胞色素c的mRNA表达(P<0.05),并降低凋亡抑制基因Bcl-2的mRNA表达(P<0.05)。荧光探针检测结果显示去甲斑蝥素可刺激细胞内ROS的产生,双重加热法检测结果显示去甲斑蝥素可诱导细胞内丙二醛(MDA)的升高(P<0.05),且呈浓度依赖性。结论去甲斑蝥素通过诱导人黑色素瘤M14细胞凋亡从而抑制其生长,其作用机制可能与激活促凋亡基因Caspase-3、Capase-9、Bax、细胞色素c,抑制抗凋亡基因Bcl-2,并诱发细胞内高ROS有关。Objective To investigate the norcantharidin induced apoptosis of melanoma M14 cells and its action mechanism.Methods The effects of different concentrations of norcantharidin on the proliferation of melanoma M14 cells were detected by CCK8 assay.The mRNA expressions of caspase-3,capase-9,Bax,Bcl-2,and cytochrome c were detected by real-time fluorescence semi-quantitative PCR(RT-PCR)method.The effects of different concentrations of norcantharidin on the production of reactive oxygen species(ROS)in cells was examined by using 2',7'-diacetyldichlorofluorescein(DCFH-DA)as a fluorescent probe,and its effects on malondialdehyde(MDA)levels in cells were detected by double heating method.Results The CCK8 assay results showed that norcantharidin inhibited the proliferation and induced the apoptosis of melanoma M14 cells in a concentration-dependent manner.RT-PCR examination results showed that norcantharidin inhibited the growth of human melanoma M14 cells by inducing its apoptosis,and the mRNA expression levels of caspase-3,capase-9,Bax and cytochrome c were gradually increased,with the increase of concentration of norcantharidin,moreover,which inhibited the mRNA expression of apoptosis-suppressing gene Bcl-2.Fluorescent probe detection results showed that norcantharidin stimulated intracellular ROS production in a concentration-dependent manner.The double heating assay showed that the levels of malondialdehyde(MDA)were increased gradually with the increase of drug concentration.Conclusion Norcantharidin can inhibit the growth of human melanoma M14 cells by inducing apoptosis,and its action mechanism may be related with the activation of proapoptotic genes caspase-3,capase-9,Bax,cytochrome c,and with the inhibition of anti-apoptosis gene Bcl-2 and with the induction of high concentration intracellular ROS.

关 键 词：黑色素瘤 去甲斑蝥素 细胞凋亡 活性氧 

分 类 号：R739.5[医药卫生—肿瘤]