TTF1-NP诱导人肝癌HepG-2细胞凋亡的内质网应激作用

doi:10.13481/j.1671-587x.20150604

摘要/Abstract

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200 μmol·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。

关键词: 珍珠梅黄酮纳米粒, 内质网应激, 细胞凋亡, 肝肿瘤

Abstract:

Objective To explore the effects of different doses of 5,2',4'-trihydroxy-6,7,5'-trimethoxyflavone nanoparticles (TTF1-NP) on theapoptosis 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 groupswere 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.

Key words: 5,2',4'-trihydroxy-6,7,5'-trimethoxyflavone nanoparticles, endoplasmic reticulum stress, apoptosis, liver neoplasms

中图分类号:

R735.7

肖斌, 刘荣荣, 刘炳彤, 张学武. TTF1-NP诱导人肝癌HepG-2细胞凋亡的内质网应激作用[J]. 吉林大学学报(医学版), 2015, 41(06): 1118-1123.

XIAO Bin, LIU Rongrong, LIU Bingtong, ZHANG Xuewu. Induction effect of TTF1-NP on human hepatoma cell apoptosis through ERS-mediated pathway[J]. Journal of Jilin University Medicine Edition, 2015, 41(06): 1118-1123.

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