摘要
目的:通过不同剂量长白山珍珠梅黄酮纳米粒(TTF1-NP)分别诱导不同种人肝癌细胞和人正常肝细胞凋亡,探讨TTF1-NP对不同细胞的作用及涉及的内质网应激作用机制。方法:以体外培养的不同种人肝癌细胞(Hep3B、HepG-2和PLC/PRF/5)和人肝细胞(Chang Liver)为模型,实验分为阴性对照组、阳性对照组(5-Fu)和TTF1-NP实验组,TTF1-NP处理浓度分别为50、100和200μmol·L-1。采用MTT法检测TTF1-NP对不同种细胞的生长抑制作用,选择最佳抑制效果细胞(HepG-2)为主要研究细胞株;利用流式细胞术检测细胞凋亡率;应用Western blotting和免疫细胞化学染色技术检测内质网应激关键蛋白表达情况;通过内质网应激抑制剂4-苯丁酸(4-PBA)作用,再次检测相关蛋白的表达情况。结果:与阴性对照组比较,不同浓度的TTF1-NP组4种细胞生长抑制率升高(P<0.05或P<0.01),且呈一定的浓度和时间依赖关系。细胞凋亡检测,与阴性对照组比较,TTF1-NP实验组随药物浓度增加其细胞凋亡率逐渐上升(P<0.05或P<0.01);内质网应激关键蛋白GRP78和caspase-4随着TTF1-NP浓度增加,表达水平逐渐升高(P<0.05或P<0.01)。4-PBA有效抑制GRP78和caspase-4表达,与TTF1-NP组比较差异有统计学意义(P<0.05或P<0.01)。结论:TTF1-NP可诱导人肝癌HepG-2细胞凋亡;内质网应激途径是TTF1-NP诱导人肝癌HepG-2细胞凋亡的主要作用机制之一。
Objective To explore the effects of different doses of 5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone nanoparticles （TTF1-NP）on the apoptosis of human hepatoma cells and human normal hepatocytes,and to explore their mechanisms through endoplasmic reticulum stress （ERS）-meditated apoptosis pathway. Methods The human hepatoma cell lines （HepG2,Hep3B and PLC/PRF/5）and human hepatocytes （Chang Liver）were used as cell model, and divided into vehicle, 5-Fu and TTF1-NP treated groups with the concentrations of 50, 100 and 200 μmol·L-1 respectively. The inhibitory effects of TTF1-NP on the cell growth were assessed using MTT assay and the best inhibitory one （HepG-2）was selected as the main research cell lines.Flow cytometry was used to detect the TTF1-NP-induced apoptosis;Western blotting and immunocytochemistry were used to determine the expressions of ERS key proteins.Finally,the expressions of key proteins were detected by Western blotting after using the ERS inhibitor 4-PBA.Results Compared with vehicle group,the inhibitory rates of growth of 4 kinds of human hepatoma cells in different concentrations of TTF1-NP groups were increased （P 〈0.05 or P 〈0.01）;moreover,the inhibitory effects of TTF1-NP were in a time-and dose-dependent manner.Compared with vehicle group,the apoptotic rates of the cells in TTF1-NP groups were increased in a dose-dependent manner （P 〈0.05 or P 〈 0.01 ）;the expression levels of ERS key proteins GRP78 and caspase-4 were increased with the increasing of the concentration of TTF1-NP （P 〈 0.05 or P 〈 0.01）.The expression levels of ERS key proteins GRP78 and caspase-4 induced by TTF1-NP were inhibited by ERS inhibitor 4-PBA （P 〈 0.05 or P 〈 0.01 ）. Conclusion TTF1-NP can induce the apoptosis of HepG2 cells;ERS pathway plays a central role in TTF1-NP-induced apoptosis of HepG-2 cells.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2015年第6期1118-1123,I0012,共7页
Journal of Jilin University：Medicine Edition
基金
国家自然科学基金资助课题(81260655)
吉林省科技厅科技发展计划项目资助课题(201215242)
关键词
珍珠梅黄酮纳米粒
内质网应激
细胞凋亡
肝肿瘤
5,2′,4′-trihydroxy-6,7,5′-trimethoxyflavone nanoparticles
endoplasmic reticulum stress
apoptosis
liver neoplasms
