CAJ | 학술논문

目的研究钠泵抑制剂哇巴因(ouabain)和华蟾毒配基(cinobufogenin)对人肝癌HepG2细胞增殖的抑制作用及细胞周期的改变,初步分析其机制.方法以人肝癌细胞HepG2为靶细胞,MTT比色法检测哇巴因和华蟾毒配基对HepG2细胞增殖的影响;Hoechst 33342荧光染色检测细胞形态学变化;流式细胞术检测细胞周期;实时定量PCR和Western blot检测CyclinA1、CDK2、PCNA和p21~(CIP1)表达的变化.结果哇巴因和华蟾毒配基可明显抑制HepG2细胞增殖,抑制作用呈时间-浓度依赖性.荧光染色显示药物处理24 h后,细胞呈现典型的凋亡形态特征;细胞周期分析显示,实验组S期细胞比例升高,实时定量PCR和Western blot结果显示:哇巴因和华蟾毒配基可下调CyclinA1、CDK2和PCNA的表达(P<0.05),上调p21~(CIP1)的表达(P<0.05).结论钠泵抑制剂可抑制肝癌HepG2细胞的增殖,引起细胞周期S期阻滞,诱导细胞凋亡,这与其调节细胞周期相关蛋白的生成关系密切.
목적 연구납빙억제제왜파인(ouabain)화화섬독배기(cinobufogenin)대인간암HepG2세포증식적억제작용급세포주기적개변,초보분석기궤제.방법 이인간암세포HepG2위파세포,MTT비색법검측왜파인화화섬독배기대HepG2세포증식적영향;Hoechst 33342형광염색검측세포형태학변화;류식세포술검측세포주기;실시정량PCR화Western blot검측CyclinA1、CDK2、PCNA화p21~(CIP1)표체적변화.결과 왜파인화화섬독배기가명현억제HepG2세포증식,억제작용정시간-농도의뢰성.형광염색현시약물처리24 h후,세포정현전형적조망형태특정;세포주기분석현시,실험조S기세포비례승고,실시정량PCR화Western blot결과현시:왜파인화화섬독배기가하조CyclinA1、CDK2화PCNA적표체(P<0.05),상조p21~(CIP1)적표체(P<0.05).결론 납빙억제제가억제간암HepG2세포적증식,인기세포주기S기조체,유도세포조망,저여기조절세포주기상관단백적생성관계밀절.
Aim To investigate the effect of ouabain and cinobufogenin on cell proliferation,apoptosis and cell cycle on HepG2,and explore their molecular mechanism.Methods The anti-proliferative effect on HepG2 cells was determined by MTT assay.The HepG2 cells were stained with Hoechst 33342,and its morphological changes were observed under fluorescence microscope;The cell cycle was measured by flow cytometry.The Cyclin A1,CDK 2,PCNA and p21~(CIP1) expression levels of HepG2 cells treated with ouabain and cinobufogenin were dectected in mRNA and protein by Real time PCR and Western blot.Results Ouabain and cinobufogenin could inhibit cell proliferation on HepG2 cells,and the inhibitory effects were in time and dose dependent manners.The HepG2 cells treated with ouabain and cinobufogenin showed the typical morphological features of apoptosis.Cell cycle analysis showed that the S phase of HepG2 cells treated with ouabain and cinobufogenin increased significantly compared with the control group.Real-time quantitative PCR and Western blot results showed that ouabain and cinobufogenin could down-regulate Cyclin A1,CDK 2,and PCNA expressions(P<0.05)and up-regulate p21~(CIP1) expression(P<0.05).Conclusion Nα~+,K~+-ATPase inhibitor has the anti-proliferative effect on HepG2 cells and induce apoptosis and S phase arrest.These effects might be related with proteins associated with cell cycle closely.