沙蟾毒精对骨肉瘤U2OS细胞增殖、凋亡、侵袭的影响及机制  

Effects of arenobufagin on proliferation and apoptosis of osteosarcoma cell line U2OS

作　　者：方硕[1] 余铃[1] 郭卫春[1] FANG Shuo;YU Ling;GUO Weichun(Renmin Hospital of Wuhan University,Wuhan 430060,China)

机构地区：[1]武汉大学人民医院,武汉430060

出　　处：《山东医药》2020年第10期45-48,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81341078、81502575)。

摘　　要：目的观察沙蟾毒精对人骨肉瘤细胞系U2OS增殖、凋亡、侵袭的影响,并探讨其作用机制。方法以人骨肉瘤细胞系U2OS作为研究对象,采用CCK-8法检测不同浓度(0、25、50、75、100、125、150、175、200 nmol/L)沙蟾毒精对U2OS细胞增殖的影响,并筛选出合适给药浓度进行后续实验;采用Hoechst 33258染色,观察U2OS细胞凋亡的形态学变化;采用Transwell侵袭实验检测沙蟾毒精对U2OS细胞侵袭能力的影响;采用实时荧光定量PCR技术检测沙蟾毒精对U2OS细胞凋亡相关基因(Bax、caspase-3)、抗凋亡相关基因(Bcl-2)及侵袭相关基因(MMP-2、MMP-9)mRNA表达的影响。结果CCK-8细胞毒性实验表明,随着沙蟾毒精浓度升高,其对U2OS细胞的抑制率逐渐增加(P均<0.05)。Hoechst 33258染色结果显示,50、100、150 nmol/L沙蟾毒精处理细胞数量减少且出现核固缩、碎裂和凋亡小体形成。Transwell侵袭实验结果显示,随沙蟾毒精(0、50、100、150 nmol/L)浓度升高,U2OS细胞透膜数逐渐减少(P均<0.05)。与0 nmol/L相比,50、100、150 nmol/L沙蟾毒精处理细胞后Bax、caspase-3 mRNA表达增加,Bcl-2、MMP-2、MMP-9 mRNA表达减少(P均<0.05)。结论沙蟾毒精能抑制U2OS细胞增殖,诱导细胞凋亡,其机制与促进Bax、caspase-3表达和抑制Bcl-2表达有关;能抑制细胞侵袭,其机制与抑制MMP-2和MMP-9表达有关。Objective To observe the effects of arenobufagin on the proliferation,apoptosis,invasion and metastasis of human osteosarcoma cell line U2OS and to investigate its mechanism.Methods Human osteosarcoma cell line U2OS was used as the research object,and CCK-8 was used to detect the effects of different concentrations(0,25,50,75,100,125,150,175,and 200 nmol/L)of arenobufagin on U2OS cell proliferation.The appropriate drug concentration was selected for the subsequent experiments.Morphological changes of U2OS cell apoptosis were observed by Hoechst 33258 staining.Transwell invasion assay was used to detect the effect of arenobufagin on invasion and metastasis of U2OS cells.Real-time fluorescence quantitative PCR was used to detect the effects of arenobufagin on the mRNA expression levels of apoptosis-related genes(Bax,Caspase-3),anti-apoptosis-related gene(Bcl-2),and invasion-related genes(MMP-2 and MMP-9)in U2OS cells.Results The cytotoxicity test of CCK-8 showed that the inhibition rate of U2OS cells increased gradually with the increased concentrations of arenobufagin(all P<0.05).The results of Hoechst 33258 staining showed that the cells in the 50,100,150 nmol/L treatment group were reduced.Nucleopolytic,fragmentation and apoptotic corpuscles were formed.Transwell invasion assay showed that the number of U2OS cells penetrating membrane decreased gradually with the increased concentrations of arenobufagin(0,50,100,150 nmol/L)(all P<0.05).Compared with the 0 nmol/L group,the mRNA expression levels of Bax and Caspase-3 increased and the mRNA expression levels of Bcl-2,MMP-2,and MMP-9 decreased in the 50,100,and and 150 nmol/L groups(all P<0.05).Conclusions Arenobufagin could inhibit the proliferation and induce the apoptosis of human osteosarcoma cell line U2OS,and its mechanism is related to the increased expression levels of Bax,Caspase-3 and the decreased expression of Bcl-2.Arenobufagin may inhibit the invasion of U2OS cells by decreasing the expression levels of MMP-2 and MMP-9.

关 键 词：骨肉瘤 沙蟾毒精 细胞凋亡 细胞侵袭 细胞转移 

分 类 号：R738.1[医药卫生—肿瘤]